Recombinant microorganisms having a methanol elongation cycle (MEC)

ABSTRACT

Provided are microorganisms that catalyze the synthesis of chemicals and biochemicals from a methanol, methane and/or formaldehyde. Also provided are methods of generating such organisms and methods of synthesizing chemicals and biochemicals using such organisms.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/785,143, filed Mar. 14, 2013, the disclosure of which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

Metabolically-modified microorganisms and methods of producing suchorganisms are provided. Also provided are methods of producing chemicalsby contacting a suitable substrate with a metabolically-modifiedmicroorganism and enzymatic preparations of the disclosure.

BACKGROUND

Acetyl-CoA is a central metabolite key to both cell growth as well asbiosynthesis of multiple cell constituents and products, including fattyacids, amino acids, isoprenoids, and alcohols. Typically, theEmbden-Meyerhof-Parnas (EMP) pathway, the Entner-Doudoroff (ED) pathway,and their variations are used to produce acetyl-CoA from sugars throughoxidative decarboxylation of pyruvate. Similarly, the CBB, RuMP, and DHApathways incorporate C1 compounds, such as CO₂ and methanol, tosynthesize sugar-phosphates and pyruvate, which then produce acetyl-CoAthrough decarboxylation of pyruvate. Thus, in all heterotrophicorganisms and those autotrophic organisms that use thesugar-phosphate-dependent pathways for C1 incorporation, acetyl-coA isderived from oxidative decarboxylation of pyruvate, resulting in loss ofone molecule of CO₂ per molecule of pyruvate. While the EMP route toacetate and ethanol has been optimized, the CO₂ loss problem has notbeen solved due to inherent pathway limitations. Without using a CO₂fixation pathway, such as the Wood-Ljungdahl pathway or the reductiveTCA cycle, the waste CO₂ leads to a significant decrease in carbonyield. This loss of carbon has a major impact on the overall economy ofbiorefinery and the carbon efficiency of cell growth.

SUMMARY

For industrial applications, the carbon utilization pathway of thedisclosure can be used to improve carbon yield in the production offuels and chemicals derived from acetyl-CoA, such as, but not limitedto, acetate, n-butanol, isobutanol, ethanol and the like. For example,if additional reducing power such as hydrogen or formic acid isprovided, the carbon utilization pathway of the disclosure can be usedto produce compounds that are more reduced than the substrate, forexample, ethanol, 1-butanol, isoprenoids, and fatty acids from sugar.

The disclosure provides a recombinant microorganism comprising ametabolic pathway for the synthesis of acetyl phosphate from methanol,methane or formaldehyde using a pathway comprising an enzyme havingfructose-6-phosphoketolase (Fpk) activity and/orxylulose-5-phosphoketolase (Xpk) activity with an acetyl-phosphate yieldbetter than a wild-type or parental organism lacking Fpk and/or Xpk. Inone embodiment, the microorganism is a prokaryote or eukaryote. Inanother embodiment, the microorganism is yeast. In yet anotherembodiment, the microorganism is a prokaryote. In yet a furtherembodiment, the microorganism is derived from an E. coli microorganism.In yet a further embodiment, the E. coli is engineered to express aphosphoketolase. In yet another embodiment, the phosphoketolase is Fpk,Xpk or a bifunctional F/Xpk enzyme. In another embodiment of any of theforegoing embodiments, the microorganism is engineered to heterologouslyexpresses one or more of the following enzymes (a) a phosphoketolase(F/Xpk); (b) a transaldolase (Tal); (c) a transketolase (Tkt); (d) aribose-5-phosphate isomerase (Rpi); (e) a ribulose-5-phosphate epimerase(Rpe); (f) a methanol dehydrogenase (Mdh); (g) a hexulose-6-phosphatesynthase (Hps); (h) a hexulose-6-phosphate isomerase (Phi); (i) adihydroxyacetone synthase (Das); and (j) a fructose-6-phosphate aldolase(Fsa). In another embodiment, the microorganism is engineered to expressa phosphoketolase derived from Bifidobaceterium adolescentis. In afurther embodiment, the phosphoketolase comprises a sequence that is atleast 49% identical to SEQ ID NO:2 and has phosphoketolase activity.

The disclosure also provides a recombinant microorganism comprising anon-CO₂-evolving pathway that comprises synthesizing acetyl phosphateusing a recombinant metabolic pathway that metabolizes methanol,methane, or formaldehyde with carbon conservation. In one embodiment,the microorganism is a prokaryote or eukaryote. In another embodiment,the microorganism is yeast. In yet another embodiment, the microorganismis a prokaryote. In yet a further embodiment, the microorganism isderived from an E. coli microorganism. In yet a further embodiment, theE. coli is engineered to express a phosphoketolase. In yet anotherembodiment, the phosphoketolase is Fpk, Xpk or a bifunctional F/Xpkenzyme. In another embodiment of any of the foregoing embodiments, themicroorganism is engineered to heterologously expresses one or more ofthe following enzymes (a) a phosphoketolase (F/Xpk); (b) a transaldolase(Tal); (c) a transketolase (Tkt); (d) a ribose-5-phosphate isomerase(Rpi); (e) a ribulose-5-phosphate epimerase (Rpe); (f) a methanoldehydrogenase (Mdh); (g) a hexulose-6-phosphate synthase (Hps); (h) ahexulose-6-phosphate isomerase (Phi); (i) a dihydroxyacetone synthase(Das); and (j) a fructose-6-phosphate aldolase (Fsa). In anotherembodiment, the microorganism is engineered to express a phosphoketolasederived from Bifidobaceterium adolescentis. In a further embodiment, thephosphoketolase comprises a sequence that is at least 49% identical toSEQ ID NO:2 and has phosphoketolase activity.

The disclosure also provides a recombinant microorganism comprising apathway that produces acetyl-phosphate through carbon rearrangement ofE4P and/or G3P and metabolism of a carbon source selected from methane,methanol, or formaldehyde. In one embodiment, the microorganism is aprokaryote or eukaryote. In another embodiment, the microorganism isyeast. In yet another embodiment, the microorganism is a prokaryote. Inyet a further embodiment, the microorganism is derived from an E. colimicroorganism. In yet a further embodiment, the E. coli is engineered toexpress a phosphoketolase. In yet another embodiment, thephosphoketolase is Fpk, Xpk or a bifunctional F/Xpk enzyme. In anotherembodiment of any of the foregoing embodiments, the microorganism isengineered to heterologously expresses one or more of the followingenzymes (a) a phosphoketolase (F/Xpk); (b) a transaldolase (Tal); (c) atransketolase (Tkt); (d) a ribose-5-phosphate isomerase (Rpi); (e) aribulose-5-phosphate epimerase (Rpe); (f) a methanol dehydrogenase(Mdh); (g) a hexulose-6-phosphate synthase (Hps); (h) ahexulose-6-phosphate isomerase (Phi); (i) a dihydroxyacetone synthase(Das); and (j) a fructose-6-phosphate aldolase (Fsa). In anotherembodiment, the microorganism is engineered to express a phosphoketolasederived from Bifidobaceterium adolescentis. In one embodiment, thephosphoketolase comprises a sequence that is at least 49% identical toSEQ ID NO:2 and has phosphoketolase activity.

The disclosure also provides a recombinant microorganism expressingenzymes that catalyze the conversion described in (i)-(xi), wherein atleast one enzyme or the regulation of at least one enzyme that performsa conversion described in (i)-(xi) is heterologous to the microorganism:(i) the production of acetyl-phosphate and erythrose-4-phosphate (E4P)from fructose-6-phosphate and/or the production of acetyl-phosphate andglyceraldehyde 3-phosphate (G3P) from xylulose 5-phosphate; (ii) thereversible conversion of fructose-6-phosphate and E4P to sedoheptulose7-phosphate (S7P) and (G3P); (iii) the reversible conversion of S7P andG3P to ribose-5-phosphate and xylulose-5-phosphate; (iv) the reversibleconversion of ribose-5-phosphate to ribulose-5-phosphate; (v) thereversible conversion of ribulose-5-phosphate to xylulose-5-phosphate;(vi) the reversible conversion of xylulose-5-phosphate and E4P tofructose-6-phosphate and glyceraldehyde-3-phosphate; (vii) theconversion of formaldehyde and ribulose-5-phosphate toD-arabino-3-Hexulose 6-phosphate; (viii) the reversible conversion ofD-arabino-3-Hexulose 6-phosphate to fructose-6-phosphate; (ix) theconversion of formaldehyde and xylulose-5-phosphate toglyceraldehyde-3-phosphate and dihydroxyacetone; (x) the conversion ofglyceraldehyde-3-phosphate and dihydroxyacetone to fructose-6-phosphate;and (xi) the conversion of methanol and a oxidized electron acceptor toformaldehyde and a reduced electron acceptor, wherein the microorganismproduces acetyl-phosphate, or compounds derived from acetyl-phosphateusing a carbon source selected from the group consisting of methanol,methane, and formaldehyde and any combination thereof. In oneembodiment, the microorganism is a prokaryote or eukaryote. In anotherembodiment, the microorganism is yeast. In another embodiment, themicroorganism is a prokaryote. In yet a further embodiment, themicroorganism is derived from an E. coli microorganism. In yet a furtherembodiment, the E. coli is engineered to express a phosphoketolase. Inanother embodiment, the phosphoketolase is Fpk, Xpk or a bifunctionalF/Xpk enzyme. In another embodiment of any of the foregoing embodiments,the microorganism is engineered to heterologously expresses one or moreof the following enzymes (a) a phosphoketolase (F/Xpk); (b) atransaldolase (Tal); (c) a transketolase (Tkt); (d) a ribose-5-phosphateisomerase (Rpi); (e) a ribulose-5-phosphate epimerase (Rpe); (f) amethanol dehydrogenase (Mdh); (g) a hexulose-6-phosphate synthase (Hps);(h) a hexulose-6-phosphate isomerase (Phi); (i) a dihydroxyacetonesynthase (Das); and (j) a fructose-6-phosphate aldolase (Fsa). Inanother embodiment, the microorganism is engineered to express aphosphoketolase derived from Bifidobaceterium adolescentis. In a furtherembodiment, the phosphoketolase comprises a sequence that is at least49% identical to SEQ ID NO:2 and has phosphoketolase activity.

The disclosure also provides a recombinant E. coli that producesacetyl-phosphate comprising expression of mdh, act, hps, phi, and f/xpk.In a further embodiment, the microorganism comprises expression of atoB,hbd, crt, ter, and adhE2, and wherein the E. coli produces 1-butanol. Inyet a further embodiment, the E. coli further comprises pta. In still afurther embodiment, the E. coli further comprises one or more knockoutsselected from the group consisting of: ΔgapA, ΔldhA, ΔfrdABCD, ΔadhE,and Δack.

The disclosure also provides a recombinant yeast that producesacetyl-phosphate comprising expression of mdh, act, hps, phi, and f/xpk.In a further embodiment, the yeast further expresses atoB, hbd, crt,ter, and adhE2, and wherein the yeast produces 1-butanol. In yet afurther embodiment, the yeast further expresses pta. In yet a furtherembodiment, the yeast further comprises one or more knockouts selectedfrom the group consisting of: Δpdc, Δadh, ΔgapA, and a glyceroldehydrogenase.

The disclosure also provides a recombinant Bacillus methanolicus thatproduces acetyl-phosphate comprising expression of f/xpk. In a furtherembodiment, the Bacillus methanolicus further expresses atoB, hbd, crt,ter, and adhE2, and wherein the Bacillus methanolicus produces1-butanol. In yet a further embodiment, the Bacillus methanolicusfurther expresses pta. In yet a further embodiment, the recombinantBacillus methanolicus further comprises one or more knockouts selectedfrom the group consisting of: Δack, and an acetaldehyde dehydrogenase(acetylating).

The disclosure also provides a recombinant microorganism comprising ametabolic pathway for the synthesis of acetyl phosphate from methanol,methane or formaldehyde using a pathway comprising an enzyme havingfructose-6-phosphoketolase (Fpk) activity and/orxylulose-5-phosphoketolase (Xpk) activity and wherein the microorganismproduces a metabolite selected from the group consisting of citrate,isocitrate, alpha-ketoglutarate, glutamate and any combination thereof.

In one embodiment, the microorganism is a prokaryote or eukaryote. Inanother embodiment, the microorganism is yeast. In yet anotherembodiment, the microorganism is a prokaryote. In yet a furtherembodiment, the microorganism is derived from an E. coli microorganism.In yet a further embodiment, the E. coli is engineered to express aphosphoketolase. In yet another embodiment, the phosphoketolase is Fpk,Xpk or a bifunctional F/Xpk enzyme. In another embodiment, themicroorganism is engineered to express a phosphoketolase derived fromBifidobaceterium adolescentis. In a further embodiment, thephosphoketolase comprises a sequence that is at least 49% identical toSEQ ID NO:2 and has phosphoketolase activity. In yet another embodiment,the recombinant microorganism is engineered to heterologously expressesone or more of the following enzymes: (a) a phosphoketolase (F/Xpk); (b)a transaldolase (Tal); (c) a transketolase (Tkt); (d) aribose-5-phosphate isomerase (Rpi); (e) a ribulose-5-phosphate epimerase(Rpe); (f) a methanol dehydrogenase (Mdh); (g) a hexulose-6-phosphatesynthase (Hps); (h) a hexulose-6-phosphate isomerase (Phi); (i) adihydroxyacetone synthase (Das); (j) a fructose-6-phosphate aldolase(Fsa); (k) a phosphoenolpyruvate carboxylase (Ppc); (l) an alcoholdehydrogenase (AdhA); (m) a phosphotransacetylase (Pta); (n) anisocitrate dehydrogenase (Icd); (o) a citrate synthase (GltA); and (p)an aconitase (Acn).

The disclosure also provides a recombinant microorganism of any of theforegoing embodiments, wherein the microorganism comprises anacetyl-phosphate (AcP) yield from a C1 carbon source (e.g., methanol,methane or formaldehyde better than 3:1, 2.9:1, 2.8:1, 2.7:1, 2.6:1,2.5:1 to about 2:1 (C1 carbon source to AcP).

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thedisclosure and, together with the detailed description, serve to explainthe principles and implementations of the invention.

FIG. 1A-D shows four variations of methanol elongation cycle (MEC)converting methanol to acetyl-phosphate (AcP) and then further to1-butanol. (a) MEC involving hps and phi activity. (b) MEC involving dasand fsa activity. (c) MEC involving hps, phi and an xpk/tkt activity.(d) MEC involving a das, fsa, and xpk/tkt activity. Other abbreviationsare: F6P, fructose 6-phosphate; E4P: erythrose-4-phosphate; G3P,glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; X5P,xylulose 5-phosphate; R5P, ribose 5-phosphate; Ru5P, ribulose5-phosphate; S7P, sedoheptulose 7-phosphate; Tal, transaldolase, Tkt,transketolase; Rpi, ribose-5-phosphate isomerase; Rpe:ribulose-5-phosphate 3-epimerase; Fpk, Fructose-6-phosphoketolase; Xpk,Xylulose-5-phosphoketolase; Mdh, Methanol dehydrogenase; Hps,hexulose-6-phosphate synthase; Phi, Hexulose-6-phosphate isomerase; Das,dihydroxyacetone synthase; Fsa, Fructose-6-phosphate aldolase; Pta,Phosphotransacetylase; and Ack, acetate kinase.

FIG. 2 shows pathways depicting formaldehyde assimilation. Formaldehydeassimilation to F6P can be accomplished by the RuMP enzymes: hps andphi. It can also be accomplished using a modified version of the DHApathway; das and fsa can also convert a pentose phosphate andformaldehyde to F6P. Phosphoketolase are well known to be able to haveX5P or dual F6P/X5P activity. When combined with transketolase, thesetwo variants of phosphoketolase are logically identical.

FIG. 3 is a graph depicting the thermodynamics of MEC. The initialoxidation of methanol to formaldehyde provides the main thermodynamichurdle. However, the core portion of MEC (the conversion of twoformaldehydes to AcCoA) is very thermodynamically favorable. The finalsequential reduction of acetyl-CoA to ethanol is also thermodynamicallyfavorable. Values were generated using the eQuilibrator website usingpH=7.5 and ionic strength at 0.2 Molar.

FIG. 4 shows and assay for methanol oxidation. The methanoldehydrogenase gene form Bacillus methanolicus is known to be activatedby a specific activator production (termed Act). Here, the same enzymecan catalyze the oxidation of methanol and the reduction of formaldehydeusing NAD or NADH, respectively. The oxidation of methanol is muchslower than the reverse direction, and is driven by large concentrationof substrate (500 mM methanol) and quick elimination of product(formaldehyde).

FIG. 5 is a graph showing the in vitro conversion of 2 Formaldehydes toAcetyl-Phosphate. The in vitro conversion of formaldehyde toacetyl-phosphate using the MEC enzymes was measured by the hydroxamatemethod. An initial amount of R5P was added to start the cycle, withexcess amounts of formaldehyde. When all the MEC enzymes were added, asignificantly higher conversion to acetyl-phosphate was achieved ascompared with the controls.

FIGS. 6-10 shows various coding sequences for enzymes useful in themethods and compositions of the disclosure.

FIGS. 11-12 show general schemes depicting MEC and additional productsthat can be formed following production of acetyl-phosphate by the MECpathway.

FIG. 13 shows a pathway for the production of citrate, isocitrate andglutamate using acetyl-phosphate produced through the MEC pathway of thedisclosure.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a polynucleotide”includes a plurality of such polynucleotides and reference to “themicroorganism” includes reference to one or more microorganisms, and soforth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Any publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

Methylotrophs are microorganisms capable of assimilating methanol intohigher carbon molecules essential for cellular growth, such asacetyl-CoA. In the known methylotrophic pathways, methanol is firstoxidized to formaldehyde. Formaldehyde can them be assimilated by one ofseveral possible routes such as the RuMP, DHA, or serine pathway. Thesepathways allow formaldehyde to be converted to sugar-phosphates orpyruvate, which can then feed into central metabolism. However, in thenative conversion of methanol to acetyl-CoA, carbon dioxide is alwaysinevitably lost during the decarboxylation of pyruvate.

The disclosure provides methods and compositions to avoid this problemin carbon management, by using a recombinant metabolic pathway to bypasspyruvate oxidation to stoichiometrically convert two methanols intoacetyl-CoA. This pathway, termed Methanol Elongation Cycle (MEC), isable to condense two methanol molecules to acetyl-CoA via a series ofwell-established enzymes. The acetyl-CoA can then be used in a number ofpathways, such as the production of bio-alcohol. In the case of1-butanol production from methanol, the overall pathway isthermodynamically favorable, ATP-independent, and redox balanced. Such aconversion has not been reported before. This pathway represents at 50%improvement in carbon balance over existing pathways and can be used inthe conversion of methanol to higher-chain liquid fuels.

The disclosure provides methods and compositions (including cell freesystems and recombinant organisms) that provide improved carbon yieldcompared to naturally occurring methanol utilization pathways. By“improved carbon yield” means that the process results in a conversionof methane, methanol, or formaldehyde to acetyl-phosphate with minimalto no carbon loss (e.g., loss as CO₂).

It should be recognized that the disclosure describes the pathway invarious embodiments and is schematically depicted in FIG. 1. It will befurther recognized the oxidative metabolism may occur after productionof acetyl-phosphate of FIG. 1.

In the pathways shown (in FIG. 1), methanol is the input molecule;however, methane and formaldehyde (among others) may also be used in thepathway. A methanol dehydrogenase is used to initiate the metabolism ofmethanol to acetyl-phosphate. The pathway uses investment of 5 carbonsugar phosphates such as, for example, ribulose-5-phosphate andxylulose-5-phosphate, which reacts with CH₂O to begin a series ofreactions involved in non-oxidative carbon rearrangement to regeneratethe 5-carbon sugar phosphates and produced acetyl-phosphate. MEC canproceed with a fructose-6-phosphoketolase (Fpk), axylulose-5-phosphoketolase (Xpk) or bifunctional enzymes that containboth activities. Because of the flexibility of MEC, the pathway canproceed with different combinations of Fpk or Xpk and Tkt, or withdifferent sugar phosphates as the starting molecule. In all thesepathways, MEC converts the combination of sugar phosphates and methanol,methane or formaldehyde to AcP without or with minimal carbon loss. AcPcan then be converted to acetyl-CoA by acetyltransferase (Pta, Ptavariant or homolog thereof), or to acetate by acetate kinase (Ack, Ackvariant or homolog thereof). Acetyl-CoA can be converted to alcohols,fatty acids, or other products if additional reducing power is provided.When producing acetyl phosphate from methanol, the MEC pathway converts4 methanol to 2 acetyl phosphates.

The disclosure provides an in vitro method of producingacetyl-phosphate, acetyl-CoA and chemicals and biofuels that useacetyl-CoA as a substrate. In this embodiment, of the disclosurecell-free preparations can be made through, for example, three methods.In one embodiment, the enzymes of the MEC pathway, as described morefully below, are purchased and mixed in a suitable buffer and a suitablesubstrate is added and incubated under conditions suitable foracetyl-phosphate production. In another embodiment, one or morepolynucleotides encoding one or more enzymes of the MEC pathway arecloned into one or more microorganism under conditions whereby theenzymes are expressed. Subsequently the cells are lysed and the lysedpreparation comprising the one or more enzymes derived from the cell arecombined with a suitable buffer and substrate (and one or moreadditional enzymes of the MEC pathway, if needed) to producedacetyl-phosphate from the substrate. Alternatively, the enzymes can beisolated from the lysed preparations and then recombined in anappropriate buffer. In yet another embodiment, a combination ofpurchased enzymes and expressed enzymes are used to provide a MECpathway in an appropriate buffer.

For example, to construct an in vitro system, all the MEC enzymes can beacquired commercially or purified by affinity chromatography, tested foractivity, and mixed together in a properly selected reaction buffer. Thesystem is ATP- and redox-independent and comprises 6 enzymatic stepsthat include the following enzymes: (i) an Mdh (methanol dehydrogenase);(ii) an Hps (hexulose-6-phosphate synthase and a Das (dihydroxyacetonesynthase) or a Phi (hexulose-6-phosphate isomerase and Fsa(fructose-6-phosphate aldolase; (iii) an Fpk (fructose-6-phosphatephosphoketolase or a Xpk (xylulose-6-phosphate phosphoketolase) and aTkt (transketolase); (iv) an Rpi (ribose-5-phosphate isomerase); (v) aTkt (transketolase); and (vi) a Tal (transaldolase).

Using this in vitro system comprising the foregoing 6 enzymatic steps aninitial amount of 4 moles of methanol can be converted to 2 moles of AcP(within error) at room temperature after about 1.5 hours.

The disclosure also provides recombinant organisms comprisingmetabolically engineered biosynthetic pathways that comprise a non-CO₂ATP independent pathway for the production of acetyl-phosphate,acetyl-CoA and/or products derived therefrom.

In one embodiment, the disclosure provides a recombinant microorganismcomprising elevated expression of at least one target enzyme as comparedto a parental microorganism or encodes an enzyme not found in theparental organism. In another or further embodiment, the microorganismcomprises a reduction, disruption or knockout of at least one geneencoding an enzyme that competes with a metabolite necessary for theproduction of a desired metabolite or which produces an unwantedproduct. The recombinant microorganism produces at least one metaboliteinvolved in a biosynthetic pathway for the production of, for example,acetyl-phosphate and/or acetyl-CoA. In general, the recombinantmicroorganisms comprises at least one recombinant metabolic pathway thatcomprises a target enzyme and may further include a reduction inactivity or expression of an enzyme in a competitive biosyntheticpathway. The pathway acts to modify a substrate or metabolicintermediate in the production of, for example, acetyl-phosphate and/oracetyl-CoA. The target enzyme is encoded by, and expressed from, apolynucleotide derived from a suitable biological source. In someembodiments, the polynucleotide comprises a gene derived from abacterial or yeast source and recombinantly engineered into themicroorganism of the disclosure.

In another embodiment, the polynucleotide encoding the desired targetenzyme is naturally occurring in the organism but is recombinantlyengineered to be overexpressed compared to the naturally expressionlevels.

As used herein, an “activity” of an enzyme is a measure of its abilityto catalyze a reaction resulting in a metabolite, i.e., to “function”,and may be expressed as the rate at which the metabolite of the reactionis produced. For example, enzyme activity can be represented as theamount of metabolite produced per unit of time or per unit of enzyme(e.g., concentration or weight), or in terms of affinity or dissociationconstants.

The term “biosynthetic pathway”, also referred to as “metabolicpathway”, refers to a set of anabolic or catabolic biochemical reactionsfor converting (transmuting) one chemical species into another. Geneproducts belong to the same “metabolic pathway” if they, in parallel orin series, act on the same substrate, produce the same product, or acton or produce a metabolic intermediate (i.e., metabolite) between thesame substrate and metabolite end product. The disclosure providesrecombinant microorganism having a metabolically engineered pathway forthe production of a desired product or intermediate.

Accordingly, metabolically “engineered” or “modified” microorganisms areproduced via the introduction of genetic material into a host orparental microorganism of choice thereby modifying or altering thecellular physiology and biochemistry of the microorganism. Through theintroduction of genetic material the parental microorganism acquires newproperties, e.g. the ability to produce a new, or greater quantities of,an intracellular metabolite. In an illustrative embodiment, theintroduction of genetic material into a parental microorganism resultsin a new or modified ability to produce acetyl-phosphate and/oracetyl-CoA through a non-CO₂ evolving and/or non-oxidative pathway foroptimal carbon utilization. The genetic material introduced into theparental microorganism contains gene(s), or parts of gene(s), coding forone or more of the enzymes involved in a biosynthetic pathway for theproduction of acetyl-phosphate and/or acetyl-CoA, and may also includeadditional elements for the expression and/or regulation of expressionof these genes, e.g. promoter sequences.

An engineered or modified microorganism can also include in thealternative or in addition to the introduction of a genetic materialinto a host or parental microorganism, the disruption, deletion orknocking out of a gene or polynucleotide to alter the cellularphysiology and biochemistry of the microorganism. Through the reduction,disruption or knocking out of a gene or polynucleotide the microorganismacquires new or improved properties (e.g., the ability to produce a newor greater quantities of an intracellular metabolite, improve the fluxof a metabolite down a desired pathway, and/or reduce the production ofundesirable by-products).

An “enzyme” means any substance, typically composed wholly or largely ofamino acids making up a protein or polypeptide that catalyzes orpromotes, more or less specifically, one or more chemical or biochemicalreactions.

As used herein, the term “metabolically engineered” or “metabolicengineering” involves rational pathway design and assembly ofbiosynthetic genes, genes associated with operons, and control elementsof such polynucleotides, for the production of a desired metabolite,such as an acetyl-phosphate and/or acetyl-CoA, higher alcohols or otherchemical, in a microorganism. “Metabolically engineered” can furtherinclude optimization of metabolic flux by regulation and optimization oftranscription, translation, protein stability and protein functionalityusing genetic engineering and appropriate culture condition includingthe reduction of, disruption, or knocking out of, a competing metabolicpathway that competes with an intermediate leading to a desired pathway.A biosynthetic gene can be heterologous to the host microorganism,either by virtue of being foreign to the host, or being modified bymutagenesis, recombination, and/or association with a heterologousexpression control sequence in an endogenous host cell. In oneembodiment, where the polynucleotide is xenogenetic to the hostorganism, the polynucleotide can be codon optimized.

A “metabolite” refers to any substance produced by metabolism or asubstance necessary for or taking part in a particular metabolic processthat gives rise to a desired metabolite, chemical, alcohol or ketone. Ametabolite can be an organic compound that is a starting material (e.g.,methanol, methane, formaldehyde etc.), an intermediate in (e.g.,acetyl-coA), or an end product (e.g., 1-butanol) of metabolism.Metabolites can be used to construct more complex molecules, or they canbe broken down into simpler ones. Intermediate metabolites may besynthesized from other metabolites, perhaps used to make more complexsubstances, or broken down into simpler compounds, often with therelease of chemical energy.

A “native” or “wild-type” protein, enzyme, polynucleotide, gene, orcell, means a protein, enzyme, polynucleotide, gene, or cell that occursin nature.

A “parental microorganism” refers to a cell used to generate arecombinant microorganism. The term “parental microorganism” describes acell that occurs in nature, i.e. a “wild-type” cell that has not beengenetically modified. The term “parental microorganism” also describes acell that serves as the “parent” for further engineering. For example, awild-type microorganism can be genetically modified to express or overexpress a first target enzyme such as a phosphoketolase. Thismicroorganism can act as a parental microorganism in the generation of amicroorganism modified to express or over-express a second target enzymee.g., a transaldolase. In turn, the microorganism can be modified toexpress or over express e.g., a transketolase and a ribose-5 phosphateisomerase, which can be further modified to express or over express athird target enzyme, e.g., a ribulose-5-phosphate epimerase.

Accordingly, a parental microorganism functions as a reference cell forsuccessive genetic modification events. Each modification event can beaccomplished by introducing one or more nucleic acid molecules in to thereference cell. The introduction facilitates the expression orover-expression of one or more target enzyme or the reduction orelimination of one or more target enzymes. It is understood that theterm “facilitates” encompasses the activation of endogenouspolynucleotides encoding a target enzyme through genetic modification ofe.g., a promoter sequence in a parental microorganism. It is furtherunderstood that the term “facilitates” encompasses the introduction ofexogenous polynucleotides encoding a target enzyme in to a parentalmicroorganism.

A “protein” or “polypeptide”, which terms are used interchangeablyherein, comprises one or more chains of chemical building blocks calledamino acids that are linked together by chemical bonds called peptidebonds. A protein or polypeptide can function as an enzyme.

Polynucleotides that encode enzymes useful for generating metabolites(e.g., enzymes such as phosphoketolase, transaldolase, transketolase,ribose-5-phosphate isomerase, ribulose-5-phosphate epimerase) includinghomologs, variants, fragments, related fusion proteins, or functionalequivalents thereof, are used in recombinant nucleic acid molecules thatdirect the expression of such polypeptides in appropriate host cells,such as bacterial or yeast cells. FIGS. 6-10 provide exemplarypolynucleotide sequences encoding polypeptides useful in the methodsdescribed herein. It is understood that the addition of sequences whichdo not alter the encoded activity of a nucleic acid molecule, such asthe addition of a non-functional or non-coding sequence, is aconservative variation of the basic nucleic acid.

It is understood that a polynucleotide described above include “genes”and that the nucleic acid molecules described above include “vectors” or“plasmids.” For example, a polynucleotide encoding a phosphoketolase cancomprise an Fpk gene or homolog thereof, or an Xpk gene or homologthereof, or a bifunctional F/Xpk gene or homolog thereof. Accordingly,the term “gene”, also called a “structural gene” refers to apolynucleotide that codes for a particular polypeptide comprising asequence of amino acids, which comprise all or part of one or moreproteins or enzymes, and may include regulatory (non-transcribed) DNAsequences, such as promoter region or expression control elements, whichdetermine, for example, the conditions under which the gene isexpressed. The transcribed region of the gene may include untranslatedregions, including introns, 5′-untranslated region (UTR), and 3′-UTR, aswell as the coding sequence.

The term “polynucleotide,” “nucleic acid” or “recombinant nucleic acid”refers to polynucleotides such as deoxyribonucleic acid (DNA), and,where appropriate, ribonucleic acid (RNA).

The term “expression” with respect to a gene or polynucleotide refers totranscription of the gene or polynucleotide and, as appropriate,translation of the resulting mRNA transcript to a protein orpolypeptide. Thus, as will be clear from the context, expression of aprotein or polypeptide results from transcription and translation of theopen reading frame.

Those of skill in the art will recognize that, due to the degeneratenature of the genetic code, a variety of codons differing in theirnucleotide sequences can be used to encode a given amino acid. Aparticular polynucleotide or gene sequence encoding a biosyntheticenzyme or polypeptide described above are referenced herein merely toillustrate an embodiment of the disclosure, and the disclosure includespolynucleotides of any sequence that encode a polypeptide comprising thesame amino acid sequence of the polypeptides and proteins of the enzymesutilized in the methods of the disclosure. In similar fashion, apolypeptide can typically tolerate one or more amino acid substitutions,deletions, and insertions in its amino acid sequence without loss orsignificant loss of a desired activity. The disclosure includes suchpolypeptides with alternate amino acid sequences, and the amino acidsequences encoded by the DNA sequences shown herein merely illustratepreferred embodiments of the disclosure.

The disclosure provides polynucleotides in the form of recombinant DNAexpression vectors or plasmids, as described in more detail elsewhereherein, that encode one or more target enzymes. Generally, such vectorscan either replicate in the cytoplasm of the host microorganism orintegrate into the chromosomal DNA of the host microorganism. In eithercase, the vector can be a stable vector (i.e., the vector remainspresent over many cell divisions, even if only with selective pressure)or a transient vector (i.e., the vector is gradually lost by hostmicroorganisms with increasing numbers of cell divisions). Thedisclosure provides DNA molecules in isolated (i.e., not pure, butexisting in a preparation in an abundance and/or concentration not foundin nature) and purified (i.e., substantially free of contaminatingmaterials or substantially free of materials with which thecorresponding DNA would be found in nature) form.

A polynucleotide of the disclosure can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques and those procedures described in the Examples section below.The nucleic acid so amplified can be cloned into an appropriate vectorand characterized by DNA sequence analysis. Furthermore,oligonucleotides corresponding to nucleotide sequences can be preparedby standard synthetic techniques, e.g., using an automated DNAsynthesizer.

It is also understood that an isolated polynucleotide molecule encodinga polypeptide homologous to the enzymes described herein can be createdby introducing one or more nucleotide substitutions, additions ordeletions into the nucleotide sequence encoding the particularpolypeptide, such that one or more amino acid substitutions, additionsor deletions are introduced into the encoded protein. Mutations can beintroduced into the polynucleotide by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. In contrast tothose positions where it may be desirable to make a non-conservativeamino acid substitution, in some positions it is preferable to makeconservative amino acid substitutions.

As will be understood by those of skill in the art, it can beadvantageous to modify a coding sequence to enhance its expression in aparticular host. The genetic code is redundant with 64 possible codons,but most organisms typically use a subset of these codons. The codonsthat are utilized most often in a species are called optimal codons, andthose not utilized very often are classified as rare or low-usagecodons. Codons can be substituted to reflect the preferred codon usageof the host, a process sometimes called “codon optimization” or“controlling for species codon bias.”

Optimized coding sequences containing codons preferred by a particularprokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl.Acids Res. 17:477-508) can be prepared, for example, to increase therate of translation or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, as compared withtranscripts produced from a non-optimized sequence. Translation stopcodons can also be modified to reflect host preference. For example,typical stop codons for S. cerevisiae and mammals are UAA and UGA,respectively. The typical stop codon for monocotyledonous plants is UGA,whereas insects and E. coli commonly use UAA as the stop codon (Dalphinet al. (1996) Nucl. Acids Res. 24: 216-218). Methodology for optimizinga nucleotide sequence for expression in a plant is provided, forexample, in U.S. Pat. No. 6,015,891, and the references cited therein.

The term “recombinant microorganism” and “recombinant host cell” areused interchangeably herein and refer to microorganisms that have beengenetically modified to express or over-express endogenouspolynucleotides, or to express non-endogenous sequences, such as thoseincluded in a vector. The polynucleotide generally encodes a targetenzyme involved in a metabolic pathway for producing a desiredmetabolite as described above, but may also include protein factorsnecessary for regulation or activity or transcription. Accordingly,recombinant microorganisms described herein have been geneticallyengineered to express or over-express target enzymes not previouslyexpressed or over-expressed by a parental microorganism. It isunderstood that the terms “recombinant microorganism” and “recombinanthost cell” refer not only to the particular recombinant microorganismbut to the progeny or potential progeny of such a microorganism.

The term “substrate” or “suitable substrate” refers to any substance orcompound that is converted or meant to be converted into anothercompound by the action of an enzyme. The term includes not only a singlecompound, but also combinations of compounds, such as solutions,mixtures and other materials which contain at least one substrate, orderivatives thereof. Further, the term “substrate” encompasses not onlycompounds that provide a carbon source suitable for use as a startingmaterial, but also intermediate and end product metabolites used in apathway associated with a metabolically engineered microorganism asdescribed herein. With respect to the MEC pathway described herein, astarting material can be any suitable carbon source including, but notlimited to, methanol, methane, formaldehyde etc. Methanol, for example,can be converted to formaldehyde prior to entering the MEC pathway asset forth in FIG. 1.

“Transformation” refers to the process by which a vector is introducedinto a host cell. Transformation (or transduction, or transfection), canbe achieved by any one of a number of means including electroporation,microinjection, biolistics (or particle bombardment-mediated delivery),or agrobacterium mediated transformation.

A “vector” generally refers to a polynucleotide that can be propagatedand/or transferred between organisms, cells, or cellular components.Vectors include viruses, bacteriophage, pro-viruses, plasmids,phagemids, transposons, and artificial chromosomes such as YACs (yeastartificial chromosomes), BACs (bacterial artificial chromosomes), andPLACs (plant artificial chromosomes), and the like, that are “episomes,”that is, that replicate autonomously or can integrate into a chromosomeof a host cell. A vector can also be a naked RNA polynucleotide, a nakedDNA polynucleotide, a polynucleotide composed of both DNA and RNA withinthe same strand, a poly-lysine-conjugated DNA or RNA, apeptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like,that are not episomal in nature, or it can be an organism whichcomprises one or more of the above polynucleotide constructs such as anagrobacterium or a bacterium.

The various components of an expression vector can vary widely,depending on the intended use of the vector and the host cell(s) inwhich the vector is intended to replicate or drive expression.Expression vector components suitable for the expression of genes andmaintenance of vectors in E. coli, yeast, Streptomyces, and othercommonly used cells are widely known and commercially available. Forexample, suitable promoters for inclusion in the expression vectors ofthe disclosure include those that function in eukaryotic or prokaryotichost microorganisms. Promoters can comprise regulatory sequences thatallow for regulation of expression relative to the growth of the hostmicroorganism or that cause the expression of a gene to be turned on oroff in response to a chemical or physical stimulus. For E. coli andcertain other bacterial host cells, promoters derived from genes forbiosynthetic enzymes, antibiotic-resistance conferring enzymes, andphage proteins can be used and include, for example, the galactose,lactose (lac), maltose, tryptophan (trp), beta-lactamase (bla),bacteriophage lambda PL, and T5 promoters. In addition, syntheticpromoters, such as the tac promoter (U.S. Pat. No. 4,551,433, which isincorporated herein by reference in its entirety), can also be used. ForE. coli expression vectors, it is useful to include an E. coli origin ofreplication, such as from pUC, p1P, p1, and pBR.

Thus, recombinant expression vectors contain at least one expressionsystem, which, in turn, is composed of at least a portion of a genecoding sequences operably linked to a promoter and optionallytermination sequences that operate to effect expression of the codingsequence in compatible host cells. The host cells are modified bytransformation with the recombinant DNA expression vectors of thedisclosure to contain the expression system sequences either asextrachromosomal elements or integrated into the chromosome.

The disclosure provides methods for the heterologous expression of oneor more of the biosynthetic genes or polynucleotides involved inacetyl-phosphate synthesis, acetyl-CoA biosynthesis or other metabolitesderived therefrom and recombinant DNA expression vectors useful in themethod. Thus, included within the scope of the disclosure arerecombinant expression vectors that include such nucleic acids.

Recombinant microorganisms provided herein can express a plurality oftarget enzymes involved in pathways for the production ofacetyl-phosphate, acetyl-CoA or other metabolites derived therefrom froma suitable carbon substrate such as, for example, methanol, methane,formaldehyde and the like. The carbon source can be metabolized to, forexample, a desirable sugar phosphate that is metabolized in the MECpathway of the disclosure. Sources of methanol, methane and formaldehydeare known. Of particular interest is methane gas, which is occurs innature and is a common by-product waste degradation.

The disclosure demonstrates that the expression or over expression ofone or more heterologous polynucleotide or over-expression of one ormore native polynucleotides encoding (i) a polypeptide that catalyzesthe production of acetyl-phosphate and erythrose-4-phosphate (E4P) fromFructose-6-phosphate; (ii) a polypeptide that catalyzes the conversionof fructose-6-phosphate and E4P to sedoheptulose 7-phosphate (S7P);(iii) a polypeptide the catalyzes the conversion of S7P toribose-5-phosphate and xylulose-5-phosphate; (iv) a polypeptide thatcatalyzes the conversion of ribose-5-phosphate to ribulose-5-phosphate;(v) a polypeptide the catalyzes the conversion of ribulose-5-phosphateto xylulose-5-phosphate; (vi) a polypeptide that converts fructose1,6-biphosphate to fructose-6-phosphate; (vii) a polypeptide thatconverts ribulose-5-phosphate and formaldehyde to hexulose-6-phosphate;(viii) a polypeptide that converts hexulose-6-phosphate tofructose-6-phosphate; (ix) a polypeptide that convertsxylulose-5-phosphate and formaldehyde to dihydroxyacetone andglyceraldehyde-3-phosphate; and (x) a polypeptide that convertsdihydroxyacetone and glyceraldehyde-3-phosphate to fructose-6-phosphate.Optionally the recombinant microorganism may further include apolypeptide that converts methanol to formaldehyde; a polypeptide thatconverts acetyl-phosphate to acetyl-coA, and/or acetyl-coA to 1-butanol.For example, the disclosure demonstrates that with expression of aheterologous a Fpk/Xpk genes in Escherichia (e.g., E. coli) theproduction of acetyl-phosphate, acetyl-CoA or other metabolites derivedtherefrom can be obtained.

Microorganisms provided herein are modified to produce metabolites inquantities and utilize carbon sources more effectively compared to aparental microorganism. In particular, the recombinant microorganismcomprises a metabolic pathway for the production of acetyl-phosphatethat conserves carbon. By “conserves carbon” is meant that the metabolicpathway that converts a sugar phosphate to acetyl-phosphate has aminimal or no loss of carbon from the starting sugar phosphate to theacetyl-phosphate. For example, in one embodiment, the recombinantmicroorganism produces a stoichiometrically conserved amount of carbonproduct from the same number of carbons in the input carbon source(e.g., 2 methanol yields 1 acetyl-phosphate).

Accordingly, the disclosure provides a recombinant microorganisms thatproduce acetyl-phosphate, acetyl-CoA or other metabolites derivedtherefrom and includes the expression or elevated expression of targetenzymes such as a phosphoketolase (e.g., Fpk, Xpk, or Fpk/Xpk), atransaldolase (e.g., Tal), a transketolase (e.g., Tkt),ribose-5-phosphate isomerase (e.g., Rpi), a ribulose-5-phosphateepimerase (e.g., Rpe), a hexulose-6-phosphate synthase (e.g., Hps), ahexulose-6-phsophate isomerase (e.g., Phi), a dihydroxyacetone synthase(e.g., Das), a fructose-6-phosphate aldolase (e.g., Fsa), a methanoldehydrogenase (e.g., Mdh), or any combination thereof, as compared to aparental microorganism. In some embodiments, where an acetyl-phosphateproduct is to be further metabolized, the recombinant microorganism canexpress or over express a phosphotransacetylase (e.g., pta), andoptionally may include expression or over expression of an acetatekinase. In addition, the microorganism may include a disruption,deletion or knockout of expression of an alcohol/acetaldehydedehydrogenase that preferentially uses acetyl-coA as a substrate (e.g.adhE gene), as compared to a parental microorganism. In someembodiments, further knockouts may include knockouts in a lactatedehydrogenase (e.g., ldh) and frdBC. It will be recognized that organismthat inherently have one or more (but not all) of the foregoing enzymes,which can be utilized as a parental organism. As described more fullybelow, a microorganism of the disclosure comprising one or morerecombinant genes encoding one or more enzymes above, and may furtherinclude additional enzymes that extend the acetyl-phosphate product toacetyl-CoA, which can then be extended to produce, for example, butanol,isobutanol, 2-pentanone and the like.

Accordingly, a recombinant microorganism provided herein includes theelevated expression of at least one target enzyme, such as FpK, Xpk, orF/Xpk. In other embodiments, a recombinant microorganism can express aplurality of target enzymes involved in a pathway to produceacetyl-phosphate, acetyl-CoA or other metabolites derived therefrom asdepicted in FIG. 1 from a carbon source such as methanol, methane,formaldehyde and the like. In one embodiment, the recombinantmicroorganism comprises expression of a heterologous or over expressionof an endogenous enzyme selected from a phosphoketolase and either (i)hexulose-6-phosphate synthase and hexulose-6-phosphate isomerase, or(ii) a dihydroxyacetone synthase and a fructose-6-phosphate aldolase. Inanother embodiment, when the microorganism expresses or overexpress atransketolase (Tkt) and/or a transaldolase (Tal).

As previously noted, the target enzymes described throughout thisdisclosure generally produce metabolites. In addition, the targetenzymes described throughout this disclosure are encoded bypolynucleotides. For example, a fructose-6-phosphoketolase can beencoded by an Fpk gene, polynucleotide or homolog thereof. The Fpk genecan be derived from any biologic source that provides a suitable nucleicacid sequence encoding a suitable enzyme havingfructose-6-phosphoketolase activity.

Accordingly, in one embodiment, a recombinant microorganism providedherein includes expression of a fructose-6-phosphoketolase (Fpk) ascompared to a parental microorganism. This expression may be combinedwith the expression or over-expression with other enzymes in themetabolic pathway for the production of acetyl-phosphate, acetyl-CoA orother metabolites derived therefrom as described herein above and below.The recombinant microorganism produces a metabolite that includesacetyl-phosphate and E4P from fructose-6-phosphate. Thefructose-6-phosphoketolase can be encoded by a Fpk gene, polynucleotideor homolog thereof. The Fpk gene or polynucleotide can be derived fromBifidobacterium adolescentis.

Phosphoketolase enzymes (F/Xpk) catalyze the formation ofacetyl-phosphate and glyceraldehyde 3-phosphate or erythrose-4-phosphatefrom xylulose 5-phosphate or fructose 6-phosphate, respectively. Forexample, the Bifidobacterium adolescentis Fpk and Xpk genes or homologsthereof can be used in the methods of the disclosure.

In addition to the foregoing, the terms “phosphoketolase” or “F/Xpk”refer to proteins that are capable of catalyzing the formation ofacetyl-phosphate and glyceraldehyde 3-phosphate or erythrose-4-phosphatefrom xylulose 5-phosphate or fructose 6-phosphate, respectively, andwhich share at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, or atleast about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greatersequence similarity, as calculated by NCBI BLAST, using defaultparameters, to SEQ ID NO:2. Additional homologs include: Gardnerellavaginalis 409-05 ref|YP_003373859.1| having 91% identity to SEQ ID NO:2;Bifidobacterium breve ref|ZP_06595931.1| having 89% to SEQ ID NO:2;Cellulomonas fimi ATCC 484 YP_004452609.1 having 55% to SEQ ID NO:2;Methylomonas methanica YP_004515101.1 having 50% identity to SEQ IDNO:2; and Thermosynechococcus elongatus BP-1] NP_681976.1 having 49%identity to SEQ ID NO:2. The sequences associated with the foregoingaccession numbers are incorporated herein by reference.

In another embodiment, a recombinant microorganism provided hereinincludes elevated expression of methanol dehydrogenase (Mdh) as comparedto a parental microorganism. This expression may be combined with theexpression or over-expression with other enzymes in the metabolicpathway for the production of acetyl-phosphate, acetyl-CoA or othermetabolites derived therefrom as described herein above and below. Therecombinant microorganism produces a metabolite that includesformaldehyde from a substrate that includes methanol. The methanoldehydrogenase can be encoded by a Mdh gene, polynucleotide or homologthereof. The Mdh gene or polynucleotide can be derived from variousmicroorganisms including B. methanolicus.

In addition to the foregoing, the terms “methanol dehydrogenase” or“Mdh” refer to proteins that are capable of catalyzing the formation offormaldehyde from methanol, and which share at least about 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity, or at least about 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99% or greater sequence similarity, as calculated byNCBI BLAST, using default parameters, to SEQ ID NO:4.

In another embodiment, a recombinant microorganism provided hereinincludes elevated expression of ribulose-5-phosphate epimerase ascompared to a parental microorganism. This expression may be combinedwith the expression or over-expression with other enzymes in themetabolic pathway for the production of acetyl-phosphate, acetyl-CoA orother metabolites derived therefrom as described herein above and below.The recombinant microorganism produces a metabolite that includesxylulose 5-phosphate from a substrate that includes ribulose5-phosphate. The ribulose-5-phosphate epimerase can be encoded by a Rpegene, polynucleotide or homolog thereof. The Rpe gene or polynucleotidecan be derived from various microorganisms including E. coli.

In addition to the foregoing, the terms “ribulose 5-phosphate epimerase”or “Rpe” refer to proteins that are capable of catalyzing the formationof xylulose 5-phosphate from ribulose 5-phosphate, and which share atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or greater sequence identity, or at least about 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequencesimilarity, as calculated by NCBI BLAST, using default parameters, toSEQ ID NO:6. Additional homologs include: Shigella boydii ATCC 9905ZP_11645297.1 having 99% identity to SEQ ID NO:6; Shewanella loihicaPV-4 YP_001092350.1 having 87% identity to SEQ ID NO:6; Nitrosococcushalophilus Nc4 YP_003526253.1 having 75% identity to SEQ ID NO:6;Ralstonia eutropha JMP134 having 72% identity to SEQ ID NO:6; andSynechococcus sp. CC9605 YP_381562.1 having 51% identity to SEQ ID NO:6.The sequences associated with the foregoing accession numbers areincorporated herein by reference.

In another embodiment, a recombinant microorganism provided hereinincludes elevated expression of ribose-5-phosphate isomerase as comparedto a parental microorganism. This expression may be combined with theexpression or over-expression with other enzymes in the metabolicpathway for the production of acetyl-phosphate, acetyl-CoA or othermetabolites derived therefrom as described herein above and below. Therecombinant microorganism produces a metabolite that includesribulose-5-phosphate from a substrate that includes ribose-5-phosphate.The ribose-5-phosphate isomerase can be encoded by a Rpi gene,polynucleotide or homolog thereof. The Rpi gene or polynucleotide can bederived from various microorganisms including E. coli.

In addition to the foregoing, the terms “ribose-5-phosphate isomerase”or “Rpi” refer to proteins that are capable of catalyzing the formationof ribulose-5-phosphate from ribose 5-phosphate, and which share atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or greater sequence identity, or at least about 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequencesimilarity, as calculated by NCBI BLAST, using default parameters, toSEQ ID NO:8. Additional homologs include: Vibrio sinaloensis DSM 21326ZP_08101051.1 having 74% identity to SEQ ID NO:8; Aeromonas media WSZP_15944363.1 having 72% identity to SEQ ID NO:8; Thermosynechococcuselongatus BP-1 having 48% identity to SEQ ID NO:8; Lactobacillussuebicus KCTC 3549 ZP_09450605.1 having 42% identity to SEQ ID NO:8; andHomo sapiens AAK95569.1 having 37% identity to SEQ ID NO:8. Thesequences associated with the foregoing accession numbers areincorporated herein by reference.

In another embodiment, a recombinant microorganism provided hereinincludes elevated expression of transaldolase as compared to a parentalmicroorganism. This expression may be combined with the expression orover-expression with other enzymes in the metabolic pathway for theproduction of acetyl-phosphate, acetyl-CoA or other metabolites derivedtherefrom as described herein above and below. The recombinantmicroorganism produces a metabolite that includessedoheptulose-7-phosphate from a substrate that includeserythrose-4-phosphate and fructose-6-phosphate. The transaldolase can beencoded by a Tal gene, polynucleotide or homolog thereof. The Tal geneor polynucleotide can be derived from various microorganisms includingE. coli.

In addition to the foregoing, the terms “transaldolase” or “Tal” referto proteins that are capable of catalyzing the formation ofsedoheptulose-7-phosphate from erythrose-4-phosphate andfructose-6-phosphate, and which share at least about 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greatersequence identity, or at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99% or greater sequence similarity, as calculated by NCBIBLAST, using default parameters, to SEQ ID NO:10. Additional homologsinclude: Bifidobacterium breve DSM 20213 ZP_06596167.1 having 30%identity to SEQ ID NO:10; Homo sapiens AAC51151.1 having 67% identity toSEQ ID NO:10; Cyanothece sp. CCY0110 ZP_01731137.1 having 57% identityto SEQ ID NO:10; Ralstonia eutropha JMP134 YP_296277.2 having 57%identity to SEQ ID NO:10; and Bacillus subtilis BEST7613 NP_440132.1having 59% identity to SEQ ID NO:10. The sequences associated with theforegoing accession numbers are incorporated herein by reference.

In another embodiment, a recombinant microorganism provided hereinincludes elevated expression of transketolase as compared to a parentalmicroorganism. This expression may be combined with the expression orover-expression with other enzymes in the metabolic pathway for theproduction of acetyl-phosphate, acetyl-CoA or other metabolites derivedtherefrom as described herein above and below. The recombinantmicroorganism produces a metabolite that includes (i) ribose-5-phosphateand xylulose-5-phosphate from sedoheptulose-7-phosphate andglyceraldehyde-3-phosphate; and/or (ii) glyceraldehyde-3-phosphate andfructose-6-phosphate from xylulose-5-phosphate anderythrose-4-phosphate. The transketolase can be encoded by a Tkt gene,polynucleotide or homolog thereof. The Tkt gene or polynucleotide can bederived from various microorganisms including E. coli.

In addition to the foregoing, the terms “transketolase” or “Tkt” referto proteins that are capable of catalyzing the formation of (i)ribose-5-phosphate and xylulose-5-phosphate fromsedoheptulose-7-phosphate and glyceraldehyde-3-phosphate; and/or (ii)glyceraldehyde-3-phosphate and fructose-6-phosphate fromxylulose-5-phosphate and erythrose-4-phosphate, and which share at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or greater sequence identity, or at least about 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence similarity,as calculated by NCBI BLAST, using default parameters, to SEQ ID NO:12.Additional homologs include: Neisseria meningitidis M13399 ZP_11612112.1having 65% identity to SEQ ID NO:12; Bifidobacterium breve DSM 20213ZP_06596168.1 having 41% identity to SEQ ID NO:12; Ralstonia eutrophaJMP134 YP_297046.1 having 66% identity to SEQ ID NO:12; Synechococcuselongatus PCC 6301 YP_171693.1 having 56% identity to SEQ ID NO:12; andBacillus subtilis BEST7613 NP_440630.1 having 54% identity to SEQ IDNO:12. The sequences associated with the foregoing accession numbers areincorporated herein by reference.

In another embodiment, a recombinant microorganism provided hereinincludes elevated expression of a hexulose-6-phosphate synthase ascompared to a parental microorganism. This expression may be combinedwith the expression or over-expression with other enzymes in themetabolic pathway for the production of acetyl-phosphate, acetyl-CoA orother metabolites derived therefrom as described herein above and below.The recombinant microorganism produces a metabolite that includeshexulose-6-phosphate from formaldehyde and ribulose-6-phosphate. Thehexulose-6-phosphate synthase can be encoded by a Hps gene,polynucleotide or homolog thereof. The Hps gene or polynucleotide can bederived from various microorganisms including B. subtilis.

In addition to the foregoing, the terms “hexulose-6-phosphate synthase”or “Hps” refer to proteins that are capable of catalyzing the formationof hexulose-6-phosphate from formaldehyde and ribulose-6-phosphate, andwhich share at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, or atleast about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greatersequence similarity, as calculated by NCBI BLAST, using defaultparameters, to SEQ ID NO:14.

In another embodiment, a recombinant microorganism provided hereinincludes elevated expression of a hexulose-6-phosphate isomerase ascompared to a parental microorganism. This expression may be combinedwith the expression or over-expression with other enzymes in themetabolic pathway for the production of acetyl-phosphate, acetyl-CoA orother metabolites derived therefrom as described herein above and below.The recombinant microorganism produces a metabolite that includesfructose-6-phosphate from hexulose-6-phosphate. The hexulose-6-phosphateisomerase can be encoded by a Phi gene, polynucleotide or homologthereof. The Phi gene or polynucleotide can be derived from variousmicroorganisms including M. Flagettus.

In addition to the foregoing, the terms “hexulose-6-phosphate isomerase”or “Phi” refer to proteins that are capable of catalyzing the formationof fructose-6-phosphate from hexulose-6-phosphate, and which share atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or greater sequence identity, or at least about 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequencesimilarity, as calculated by NCBI BLAST, using default parameters, toSEQ ID NO:16.

In another embodiment, a recombinant microorganism provided hereinincludes elevated expression of a dihydroxyacetone synthase as comparedto a parental microorganism. This expression may be combined with theexpression or over-expression with other enzymes in the metabolicpathway for the production of acetyl-phosphate, acetyl-CoA or othermetabolites derived therefrom as described herein above and below. Therecombinant microorganism produces a metabolite that includesdihydroxyacetone and glyceraldehyde-3-phosphate fromxylulose-5-phosphate and formaldehyde. The dihydroxyacetone synthase canbe encoded by a Das gene, polynucleotide or homolog thereof. The Dasgene or polynucleotide can be derived from various microorganismsincluding C. boindii.

In addition to the foregoing, the terms “dihydroxyacetone synthase” or“Das” refer to proteins that are capable of catalyzing the formation ofdihydroxyacetone and glyceraldehyde-3-phosphate fromxylulose-5-phosphate and formaldehyde, and which share at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or greater sequence identity, or at least about 50%, 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence similarity, ascalculated by NCBI BLAST, using default parameters, to SEQ ID NO:18.

In another embodiment, a recombinant microorganism provided hereinincludes elevated expression of a fructose-6-phosphate aldolase ascompared to a parental microorganism. This expression may be combinedwith the expression or over-expression with other enzymes in themetabolic pathway for the production of acetyl-phosphate, acetyl-CoA orother metabolites derived therefrom as described herein above and below.The recombinant microorganism produces a metabolite that includesfructose-6-phosphate from glyceraldehyde-3-phosphate anddihydroxyacetone. The fructose-6-phosphate aldolase can be encoded by aFsa gene, polynucleotide or homolog thereof. The Fsa gene orpolynucleotide can be derived from various microorganisms including S.enterica.

In addition to the foregoing, the terms “fructose-6-phosphate aldolase”or “Fsa” refer to proteins that are capable of catalyzing the formationof fructose-6-phosphate from glyceraldehyde-3-phosphate anddihydroxyacetone, and which share at least about 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greatersequence identity, or at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99% or greater sequence similarity, as calculated by NCBIBLAST, using default parameters, to SEQ ID NO:20.

In yet another embodiment, a recombinant microorganism provided hereinincludes elevated expression of a crotonyl-CoA reductase as compared toa parental microorganism. This expression may be combined with theexpression or over-expression with other enzymes in the metabolicpathway for the production of n-butanol, isobutanol, butyryl-coA and/oracetone. The microorganism produces a metabolite that includesbutyryl-CoA from a substrate that includes crotonyl-CoA. Thecrotonyl-CoA reductase can be encoded by a ccr gene, polynucleotide orhomolog thereof. The ccr gene or polynucleotide can be derived from thegenus Streptomyces. Alternatively, or in addition to, the microorganismprovided herein includes elevated expression of a trans-2-hexenoyl-CoAreductase as compared to a parental microorganism. The microorganismproduces a metabolite that includes butyryl-CoA from a substrate thatincludes crotonyl-CoA. The trans-2-hexenoyl-CoA reductase can alsoconvert trans-2-hexenoyl-CoA to hexanoyl-CoA. The trans-2-hexenoyl-CoAreductase can be encoded by a ter gene, polynucleotide or homologthereof. The ter gene or polynucleotide can be derived from the genusEuglena. The ter gene or polynucleotide can be derived from Treponemadenticola. The enzyme from Euglena gracilis acts on crotonoyl-CoA and,more slowly, on trans-hex-2-enoyl-CoA and trans-oct-2-enoyl-CoA.

Trans-2-enoyl-CoA reductase or TER is a protein that is capable ofcatalyzing the conversion of crotonyl-CoA to butyryl-CoA, andtrans-2-hexenoyl-CoA to hexanoyl-CoA. In certain embodiments, therecombinant microorganism expresses a TER which catalyzes the samereaction as Bcd/EtfA/EtfB from Clostridia and other bacterial species.Mitochondrial TER from E. gracilis has been described, and many TERproteins and proteins with TER activity derived from a number of specieshave been identified forming a TER protein family (see, e.g., U.S. Pat.Appl. 2007/0022497 to Cirpus et al.; and Hoffmeister et al., J. Biol.Chem., 280:4329-4338, 2005, both of which are incorporated herein byreference in their entirety). A truncated cDNA of the E. gracilis genehas been functionally expressed in E. coli.

TER proteins can also be identified by generally well knownbioinformatics methods, such as BLAST. Examples of TER proteins include,but are not limited to, TERs from species such as: Euglena spp.including, but not limited to, E. gracilis, Aeromonas spp. including,but not limited, to A. hydrophila, Psychromonas spp. including, but notlimited to, P. ingrahamii, Photobacterium spp. including, but notlimited, to P. profundum, Vibrio spp. including, but not limited, to V.angustum, V. cholerae, V. alginolyticus, V. parahaemolyticus, V.vulnificus, V. fischeri, V. splendidus, Shewanella spp. including, butnot limited to, S. amazonensis, S. woodyi, S. frigidimarina, S.paeleana, S. baltica, S. denitrificans, Oceanospirillum spp.,Xanthomonas spp. including, but not limited to, X. oryzae, X.campestris, Chromohalobacter spp. including, but not limited, to C.salexigens, Idiomarina spp. including, but not limited, to I. baltica,Pseudoalteromonas spp. including, but not limited to, P. atlantica,Alteromonas spp., Saccharophagus spp. including, but not limited to, S.degradans, S. marine gamma proteobacterium, S. alpha proteobacterium,Pseudomonas spp. including, but not limited to, P. aeruginosa, P.putida, P. fluorescens, Burkholderia spp. including, but not limited to,B. phytofirmans, B. cenocepacia, B. cepacia, B. ambifaria, B.vietnamensis, B. multivorans, B. dolosa, Methylbacillus spp. including,but not limited to, M. flageliatus, Stenotrophomonas spp. including, butnot limited to, S. maltophilia, Congregibacter spp. including, but notlimited to, C. litoralis, Serratia spp. including, but not limited to,S. proteamaculans, Marinomonas spp., Xytella spp. including, but notlimited to, X. fastidiosa, Reinekea spp., Colweffia spp. including, butnot limited to, C. psychrerythraea, Yersinia spp. including, but notlimited to, Y. pestis, Y. pseudotuberculosis, Methylobacillus spp.including, but not limited to, M. flagellatus, Cytophaga spp. including,but not limited to, C. hutchinsonii, Flavobacterium spp. including, butnot limited to, F. johnsoniae, Microscilla spp. including, but notlimited to, M. marina, Polaribacter spp. including, but not limited to,P. irgensii, Clostridium spp. including, but not limited to, C.acetobutylicum, C. beijerenckii, C. cellulolyticum, Coxiella spp.including, but not limited to, C. burnetii.

In addition to the foregoing, the terms “trans-2-enoyl-CoA reductase” or“TER” refer to proteins that are capable of catalyzing the conversion ofcrotonyl-CoA to butyryl-CoA, or trans-2-hexenoyl-CoA to hexanoyl-CoA andwhich share at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, or atleast about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greatersequence similarity, as calculated by NCBI BLAST, using defaultparameters, to either or both of the truncated E. gracilis TER or thefull length A. hydrophila TER.

In yet another embodiment, a recombinant microorganism provided hereinincludes elevated expression of a butyryl-CoA dehydrogenase as comparedto a parental microorganism. This expression may be combined with theexpression or over-expression with other enzymes in the metabolicpathway for the production of 1-butanol, isobutanol, acetone, octanol,hexanol, 2-pentanone, and butyryl-coA as described herein above andbelow. The recombinant microorganism produces a metabolite that includesbutyryl-CoA from a substrate that includes crotonyl-CoA. The butyryl-CoAdehydrogenase can be encoded by a bcd gene, polynucleotide or homologthereof. The bcd gene, polynucleotide can be derived from Clostridiumacetobutylicum, Mycobacterium tuberculosis, or Megasphaera elsdenii.

In another embodiment, a recombinant microorganism provided hereinincludes expression or elevated expression of an acetyl-CoAacetyltransferase as compared to a parental microorganism. Themicroorganism produces a metabolite that includes acetoacetyl-CoA from asubstrate that includes acetyl-CoA. The acetyl-CoA acetyltransferase canbe encoded by a thlA gene, polynucleotide or homolog thereof. The thlAgene or polynucleotide can be derived from the genus Clostridium.

Pyruvate-formate lyase (Formate acetylytransferase) is an enzyme thatcatalyzes the conversion of pyruvate to acetyl)-coA and formate. It isinduced by pfl-activating enzyme under anaerobic conditions bygeneration of an organic free radical and decreases significantly duringphosphate limitation. Formate acetylytransferase is encoded in E. coliby pflB. PFLB homologs and variants are known. For examples, suchhomologs and variants include, for example, Formate acetyltransferase 1(Pyruvate formate-lyase 1) gi|129879|sp|P09373.2|PFLB_ECOLI(129879);formate acetyltransferase 1 (Yersinia pestis CO92)gi|16121663|ref|NP_404976.1|(16121663); formate acetyltransferase 1(Yersinia pseudotuberculosis IP 32953)gi|51595748|ref|YP_069939.1|(51595748); formate acetyltransferase 1(Yersinia pestis biovar Microtus str. 91001)gi|45441037|ref|NP_992576.1|(45441037); formate acetyltransferase 1(Yersinia pestis CO92) gi|115347142|emb|CAL20035.1|(115347142); formateacetyltransferase 1 (Yersinia pestis biovar Microtus str. 91001)gi|45435896|gb|AAS61453.1|(45435896); formate acetyltransferase 1(Yersinia pseudotuberculosis IP 32953)gi|51589030|emb|CAH20648.1|(51589030); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Typhi str. CT18)gi|16759843|ref|NP_455460.1|(16759843); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150)gi|56413977|ref|YP_151052.1|(56413977); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Typhi)gi|16502136|emb|CAD05373.1|(16502136); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150)gi|56128234|gb|AAV77740.1|(56128234); formate acetyltransferase 1(Shigella dysenteriae Sd197) gi|82777577|ref|YP_403926.1|(82777577);formate acetyltransferase 1 (Shigella flexneri 2a str. 2457T)gi|30062438|ref|NP_836609.1|(30062438); formate acetyltransferase 1(Shigella flexneri 2a str. 2457T) gi|30040684|gb|AAP16415.1|(30040684);formate acetyltransferase 1 (Shigella flexneri 5 str. 8401)gi|110614459|gb|ABF03126.1|(110614459); formate acetyltransferase 1(Shigella dysenteriae Sd197) gi|81241725|gb|ABB62435.1|(81241725);formate acetyltransferase 1 (Escherichia coli O157:H7 EDL933)gi|12514066|gb|AAG55388.1|AE005279_8(12514066); formateacetyltransferase 1 (Yersinia pestis KIM)gi|22126668|ref|NP_670091.1|(22126668); formate acetyltransferase 1(Streptococcus agalactiae A909) gi|76787667|ref|YP_330335.1|(76787667);formate acetyltransferase 1 (Yersinia pestis KIM)gi|21959683|gb|AAM86342.1|AE013882_3(21959683); formateacetyltransferase 1 (Streptococcus agalactiae A909)gi|76562724|gb|ABA45308.1|(76562724); formate acetyltransferase 1(Yersinia enterocolitica subsp. enterocolitica 8081)gi|123441844|ref|YP_001005827.1|(123441844); formate acetyltransferase 1(Shigella flexneri 5 str. 8401)gi|110804911|ref|YP_688431.1|(110804911); formate acetyltransferase 1(Escherichia coli UTI89) gi|91210004|ref|YP_539990.1|(91210004); formateacetyltransferase 1 (Shigella boydii Sb227)gi|82544641|ref|YP_408588.1|(82544641); formate acetyltransferase 1(Shigella sonnei Ss046) gi|74311459|ref|YP_309878.1|(74311459); formateacetyltransferase 1 (Klebsiella pneumoniae subsp. pneumoniae MGH 78578)gi|152969488|ref|YP_001334597.1|(152969488); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Typhi Ty2)gi|29142384|ref|NP_805726.1|(29142384) formate acetyltransferase 1(Shigella flexneri 2a str. 301) gi|24112311|ref|NP_706821.1|(24112311);formate acetyltransferase 1 (Escherichia coli O157:H7 EDL933)gi|15800764|ref|NP_286778.1|(15800764); formate acetyltransferase 1(Klebsiella pneumoniae subsp. pneumoniae MGH 78578)gi|150954337|gb|ABR76367.1|(150954337); formate acetyltransferase 1(Yersinia pestis CA88-4125) gi|149366640|ref|ZP_01888674.1|(149366640);formate acetyltransferase 1 (Yersinia pestis CA88-4125)gi|149291014|gb|EDM41089.1|(149291014); formate acetyltransferase 1(Yersinia enterocolitica subsp. enterocolitica 8081)gi|122088805|emb|CAL11611.1|(122088805); formate acetyltransferase 1(Shigella sonnei Ss046) gi|73854936|gb|AAZ87643.1|(73854936); formateacetyltransferase 1 (Escherichia coli UTI89)gi|91071578|gb|ABE06459.1|(91071578); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Typhi Ty2)gi|29138014|gb|AA069575.1|(29138014); formate acetyltransferase 1(Shigella boydii Sb227) gi|81246052|gb|ABB66760.1|(81246052); formateacetyltransferase 1 (Shigella flexneri 2a str. 301)gi|24051169|gb|AAN42528.1|(24051169); formate acetyltransferase 1(Escherichia coli O157:H7 str. Sakai)gi|13360445|dbj|BAB34409.1|(13360445); formate acetyltransferase 1(Escherichia coli O157:H7 str. Sakai)gi|15830240|ref|NP_309013.1|(15830240); formate acetyltransferase I(pyruvate formate-lyase 1) (Photorhabdus luminescens subsp. laumondiiTTO1) gi|36784986|emb|CAE13906.1|(36784986); formate acetyltransferase I(pyruvate formate-lyase 1) (Photorhabdus luminescens subsp. laumondiiTTO1) gi|37525558|ref|NP_928902.1|(37525558); formate acetyltransferase(Staphylococcus aureus subsp. aureus Mu50)gi|14245993|dbj|BAB56388.1|(14245993); formate acetyltransferase(Staphylococcus aureus subsp. aureus Mu50)gi|15923216|ref|NP_370750.1|(15923216); Formate acetyltransferase(Pyruvate formate-lyase) gi|81706366|sp|Q7A7X6.1|PFLB_STAAN (81706366);Formate acetyltransferase (Pyruvate formate-lyase)gi|81782287|sp|Q99WZ7.1|PFLB_STAAM(81782287); Formate acetyltransferase(Pyruvate formate-lyase) gi|81704726|sp|Q7A1W9.1|PFLB_STAAW(81704726);formate acetyltransferase (Staphylococcus aureus subsp. aureus Mu3)gi|156720691|dbj|BAF77108.1|(156720691); formate acetyltransferase(Erwinia carotovora subsp. atroseptica SCRI1043)gi|50121521|ref|YP_050688.1|(50121521); formate acetyltransferase(Erwinia carotovora subsp. atroseptica SCRI1043)gi|49612047|emb|CAG75496.1|(49612047); formate acetyltransferase(Staphylococcus aureus subsp. aureus str. Newman)gi|150373174|dbj|BAF66434.1|(150373174); formate acetyltransferase(Shewanella oneidensis MR-1) gi|24374439|ref|NP_718482.1|(24374439);formate acetyltransferase (Shewanella oneidensis MR-1)gi|24349015|gb|AAN55926.1|AE015730_3(24349015); formateacetyltransferase (Actinobacillus pleuropneumoniae serovar 3 str. JL03)gi|165976461|ref|YP_001652054.1|(165976461); formate acetyltransferase(Actinobacillus pleuropneumoniae serovar 3 str. JL03)gi|165876562|gb|ABY69610.1|(165876562); formate acetyltransferase(Staphylococcus aureus subsp. aureus MW2)gi|21203365|dbj|BAB94066.1|(21203365); formate acetyltransferase(Staphylococcus aureus subsp. aureus N315)gi|13700141|dbj|BAB41440.1|(13700141); formate acetyltransferase(Staphylococcus aureus subsp. aureus str. Newman)gi|151220374|ref|YP_001331197.1|(151220374); formate acetyltransferase(Staphylococcus aureus subsp. aureus Mu3)gi|156978556|ref|YP_001440815.1|(156978556); formate acetyltransferase(Synechococcus sp. JA-2-3B′ a(2-13))gi|86607744|ref|YP_476506.1|(86607744); formate acetyltransferase(Synechococcus sp. JA-3-3Ab) gi|86605195|ref|YP_473958.1|(86605195);formate acetyltransferase (Streptococcus pneumoniae D39)gi|116517188|ref|YP_815928.1|(116517188); formate acetyltransferase(Synechococcus sp. JA-2-3B′ a(2-13))gi|86556286|gb|ABD01243.1|(86556286); formate acetyltransferase(Synechococcus sp. JA-3-3Ab) gi|86553737|gb|ABC98695.1|(86553737);formate acetyltransferase (Clostridium novyi NT)gi|118134908|gb|ABK61952.1|(118134908); formate acetyltransferase(Staphylococcus aureus subsp. aureus MRSA252)gi|49482458|ref|YP_039682.1|(49482458); and formate acetyltransferase(Staphylococcus aureus subsp. aureus MRSA252)gi|49240587|emb|CAG39244.1|(49240587), each sequence associated with theaccession number is incorporated herein by reference.

FNR transcriptional dual regulators are transcription regulatorsresponsive to oxygen contenct. FNR is an anaerobic regulator thatrepresses the expression of PDHc. Accordingly, reducing FNR will resultin an increase in PDHc expression. FNR homologs and variants are known.For examples, such homologs and variants include, for example,DNA-binding transcriptional dual regulator, global regulator ofanaerobic growth (Escherichia coli W3110)gi|1742191|dbj|BAA14927.1|(1742191); DNA-binding transcriptional dualregulator, global regulator of anaerobic growth (Escherichia coli K12)gi|16129295|ref|NP_415850.1|(16129295); DNA-binding transcriptional dualregulator, global regulator of anaerobic growth (Escherichia coli K12)gi|1787595|gb|AAC74416.1|(1787595); DNA-binding transcriptional dualregulator, global regulator of anaerobic growth (Escherichia coli W3110)gi|89108182|ref|AP_001962.1|(89108182); fumarate/nitrate reductiontranscriptional regulator (Escherichia coli UTI89)gi|162138444|ref|YP_540614.2|(162138444); fumarate/nitrate reductiontranscriptional regulator (Escherichia coli CFT073)gi|161486234|ref|NP_753709.2|(161486234); fumarate/nitrate reductiontranscriptional regulator (Escherichia coli O157:H7 EDL933)gi|15801834|ref|NP_287852.1|(15801834); fumarate/nitrate reductiontranscriptional regulator (Escherichia coli APEC O1)gi|117623587|ref|YP_852500.1|(117623587); fumarate and nitrate reductionregulatory protein gi|71159334|sp|P0A9E5.1|FNR_ECOL6 (71159334);transcriptional regulation of aerobic, anaerobic respiration, osmoticbalance (Escherichia coli O157:H7 EDL933)gi|12515424|gb|AAG56466.1|AE005372_11(12515424); Fumarate and nitratereduction regulatory proteingi|71159333|sp|P0A9E6.1|FNR_ECOL6(71159333); Fumarate and nitratereduction Regulatory protein (Escherichia coli CFT073)gi|26108071|gb|AAN80271.1|AE016760_130(26108071); fumarate and nitratereduction regulatory protein (Escherichia coli UTI89)gi|91072202|gb|ABE07083.1|(91072202); fumarate and nitrate reductionregulatory protein (Escherichia coli HS)gi|157160845|ref|YP_001458163.1|(157160845); fumarate and nitratereduction regulatory protein (Escherichia coli E24377A)gi|157157974|ref|YP_001462642.1|(157157974); fumarate and nitratereduction regulatory protein (Escherichia coli E24377A)gi|157080004|gb|ABV19712.1|(157080004); fumarate and nitrate reductionregulatory protein (Escherichia coli HS)gi|157066525|gb|ABV05780.1|(157066525); fumarate and nitrate reductionregulatory protein (Escherichia coli APEC O1)gi|115512711|gb|ABJ00786.1|(115512711); transcription regulator Fnr(Escherichia coli O157:H7 str. Sakai)gi|13361380|dbj|BAB35338.1|(13361380) DNA-binding transcriptional dualregulator (Escherichia coli K12) gi|16131236|ref|NP_417816.1|(16131236),to name a few, each sequence associated with the accession number isincorporated herein by reference.

An acetoacetyl-coA thiolase (also sometimes referred to as an acetyl-coAacetyltransferase) catalyzes the production of acetoacetyl-coA from twomolecules of acetyl-coA. Depending upon the organism used a heterologousacetoacetyl-coA thiolase (acetyl-coA acetyltransferase) can beengineered for expression in the organism. Alternatively a nativeacetoacetyl-coA thiolase (acetyl-coA acetyltransferase) can beoverexpressed. Acetoacetyl-coA thiolase is encoded in E. coli by thl.Acetyl-coA acetyltransferase is encoded in C. acetobutylicum by atoB.THL and AtoB homologs and variants are known. For examples, suchhomologs and variants include, for example, acetyl-coa acetyltransferase(thiolase) (Streptomyces coelicolor A3(2))gi|21224359|ref|NP_630138.1|(21224359); acetyl-coa acetyltransferase(thiolase) (Streptomyces coelicolor A3(2))gi|3169041|emb|CAA19239.1|(3169041); Acetyl CoA acetyltransferase(thiolase) (Alcanivorax borkumensis SK2)gi|110834428|ref|YP_693287.1|(110834428); Acetyl CoA acetyltransferase(thiolase) (Alcanivorax borkumensis SK2)gi|110647539|emb|CAL17015.1|(110647539); acetyl CoA acetyltransferase(thiolase) (Saccharopolyspora erythraea NRRL 2338)gi|133915420|emb|CAM05533.1|(133915420); acetyl-coa acetyltransferase(thiolase) (Saccharopolyspora erythraea NRRL 2338)gi|134098403|ref|YP_001104064.1|(134098403); acetyl-coaacetyltransferase (thiolase) (Saccharopolyspora erythraea NRRL 2338)gi|133911026|emb|CAM01139.1|(133911026); acetyl-CoA acetyltransferase(thiolase) (Clostridium botulinum A str. ATCC 3502)gi|148290632|emb|CAL84761.1|(148290632); acetyl-CoA acetyltransferase(thiolase) (Pseudomonas aeruginosa UCBPP-PA14)gi|115586808|gb|ABJ12823.1|(115586808); acetyl-CoA acetyltransferase(thiolase) (Ralstonia metallidurans CH34)gi|93358270|gb|ABF12358.1|(93358270); acetyl-CoA acetyltransferase(thiolase) (Ralstonia metallidurans CH34)gi|93357190|gb|ABF11278.1|(93357190); acetyl-CoA acetyltransferase(thiolase) (Ralstonia metallidurans CH34)gi|93356587|gb|ABF10675.1|(93356587); acetyl-CoA acetyltransferase(thiolase) (Ralstonia eutropha JMP134)gi|72121949|gb|AAZ64135.1|(72121949); acetyl-CoA acetyltransferase(thiolase) (Ralstonia eutrophaJMP134)gi|72121729|gb|AAZ63915.1|(72121729); acetyl-CoAacetyltransferase (thiolase) (Ralstonia eutropha JMP134)gi|72121320|gb|AAZ63506.1|(72121320); acetyl-CoA acetyltransferase(thiolase) (Ralstonia eutropha JMP134)gi|72121001|gb|AAZ63187.1|(72121001); acetyl-CoA acetyltransferase(thiolase) (Escherichia coli) gi|2764832|emb|CAA66099.1|(2764832), eachsequence associated with the accession number is incorporated herein byreference.

Butyryl-coA dehydrogenase is an enzyme in the protein pathway thatcatalyzes the reduction of crotonyl-CoA to butyryl-CoA. A butyryl-CoAdehydrogenase complex (Bcd/EtfAB) couples the reduction of crotonyl-CoAto butyryl-CoA with the reduction of ferredoxin. Depending upon theorganism used a heterologous butyryl-CoA dehydrogenase can be engineeredfor expression in the organism. Alternatively, a native butyryl-CoAdehydrogenase can be overexpressed. Butyryl-coA dehydrognase is encodedin C. acetobuylicum and M. elsdenii by bcd. BCD homologs and variantsare known. For examples, such homologs and variants include, forexample, butyryl-CoA dehydrogenase (Clostridium acetobutylicum ATCC 824)gi|15895968|ref|NP_349317.1|(15895968); Butyryl-CoA dehydrogenase(Clostridium acetobutylicum ATCC 824)gi|15025744|gb|AAK80657.1|AE007768_11(15025744); butyryl-CoAdehydrogenase (Clostridium botulinum A str. ATCC 3502)gi|148381147|ref|YP_001255688.1|(148381147); butyryl-CoA dehydrogenase(Clostridium botulinum A str. ATCC 3502)gi|148290631|emb|CAL84760.1|(148290631), each sequence associated withthe accession number is incorporated herein by reference in itsentirety. BCD can be expressed in combination with a flavoprotienelectron transfer protein. Useful flavoprotein electron transfer proteinsubunits are expressed in C. acetobutylicum and M. elsdenii by a geneetfA and etfB (or the operon etfAB). ETFA, B, and AB homologs andvariants are known. For examples, such homologs and variants include,for example, putative a-subunit of electron-transfer flavoproteingi|1055221|gb|AAA95970.1|(1055221); putative b-subunit ofelectron-transfer flavoprotein gi|1055220|gb|AAA95969.1|(1055220), eachsequence associated with the accession number is incorporated herein byreference.

Crotonyl-coA reductase catalyzes the reduction of crotonyl-CoA tobutyryl-CoA. Depending upon the organism used a heterologousCrotonyl-coA reductase can be engineered for expression in the organism.Alternatively, a native Crotonyl-coA reductase can be overexpressed.Crotonyl-coA reductase is encoded in S. coelicolor by ccr. CCR homologsand variants are known. For examples, such homologs and variantsinclude, for example, crotonyl CoA reductase (Streptomyces coelicolorA3(2)) gi|21224777|ref|NP_630556.1|(21224777); crotonyl CoA reductase(Streptomyces coelicolor A3(2)) gi|4154068|emb|CAA22721.1|(4154068);crotonyl-CoA reductase (Methylobacterium sp. 4-46)gi|168192678|gb|ACA14625.1|(168192678); crotonyl-CoA reductase(Dinoroseobacter shibae DFL 12)gi|159045393|ref|YP_001534187.1|(159045393); crotonyl-CoA reductase(Salinispora arenicola CNS-205)gi|159039522|ref|YP_001538775.1|(159039522); crotonyl-CoA reductase(Methylobacterium extorquens PA1)gi|163849740|ref|YP_001637783.1|(163849740); crotonyl-CoA reductase(Methylobacterium extorquens PA1)gi|163661345|gb|ABY28712.1|(163661345); crotonyl-CoA reductase(Burkholderia ambifaria AMMD) gi|115360962|ref|YP_778099.1|(115360962);crotonyl-CoA reductase (Parvibaculum lavamentivorans DS-1)gi|154252073|ref|YP_001412897.1|(154252073); Crotonyl-CoA reductase(Silicibacter sp. TM1040) gi|99078082|ref|YP_611340.1|(99078082);crotonyl-CoA reductase (Xanthobacter autotrophicus Py2)gi|154245143|ref|YP_001416101.1|(154245143); crotonyl-CoA reductase(Nocardioides sp. JS614) gi|119716029|ref|YP_922994.1|(119716029);crotonyl-CoA reductase (Nocardioides sp. JS614)gi|119536690|gb|ABL81307.1|(119536690); crotonyl-CoA reductase(Salinispora arenicola CNS-205) gi|157918357|gb|ABV99784.1|(157918357);crotonyl-CoA reductase (Dinoroseobacter shibae DFL 12)gi|157913153|gb|ABV94586.1|(157913153); crotonyl-CoA reductase(Burkholderia ambifaria AMMD) gi|115286290|gb|ABI91765.1|(115286290);crotonyl-CoA reductase (Xanthobacter autotrophicus Py2)gi|154159228|gb|ABS66444.1|(154159228); crotonyl-CoA reductase(Parvibaculum lavamentivorans DS-1)gi|154156023|gb|ABS63240.1|(154156023); crotonyl-CoA reductase(Methylobacterium radiotolerans JCM 2831)gi|170654059|gb|ACB23114.1|(170654059); crotonyl-CoA reductase(Burkholderia graminis C4D1M) gi|170140183|gb|EDT08361.1|(170140183);crotonyl-CoA reductase (Methylobacterium sp. 4-46)gi|168198006|gb|ACA19953.1|(168198006); crotonyl-CoA reductase (Frankiasp. EAN1pec) gi|158315836|ref|YP_001508344.1|(158315836), each sequenceassociated with the accession number is incorporated herein by referencein its entirety.

In yet other embodiment, in addition to any of the foregoing andcombinations of the foregoing, additional genes/enzymes may be used toproduce a desired product. For example, the following table provideenzymes that can be combined with the MEC pathway enzymes for theproduction of 1-butanol:

Exemplary Exemplary Enzyme Gene(s) 1-butanol Organism EthanolDehydrogenase adhE − E. coli Lactate Dehydrogenase ldhA − E. coliFumarate reductase frdB, − E. coli frdC, or frdBC Oxygen transcriptionfnr − E. coli regulator Phosphate pta − E. coli acetyltransferaseFormate pflB − E. coli acetyltransferase acetyl-coA atoB + C.acetobutylicum acetyltransferase acetoacetyl-coA thl, thlA, + E. coli,thiolase thlB C. acetobutylicum 3-hydroxybutyryl-CoA hbd + C.acetobutylicum dehydrogenase crotonase crt + C. acetobutylicumbutyryl-CoA bcd + C. acetobutylicum, dehydrogenase M. elsdenii electrontransfer etfAB + C. acetobutylicum, flavoprotein M. elsdeniialdehyde/alcohol adhE2 + C. acetobutylicum dehydrogenase(butyral-bdhA/bdhB dehyde aad dehydrogenase/butanol dehydrogenase) crotonyl-coAreductase ccr + S. coelicolor trans-2-enoyl-CoA Ter + T. denticola,reductase F. succinogenes * knockout or a reduction in expression areoptional in the synthesis of the product, however, such knockoutsincrease various substrate intermediates and improve yield.

In addition, and as mentioned above, homologs of enzymes useful forgenerating metabolites are encompassed by the microorganisms and methodsprovided herein. The term “homologs” used with respect to an originalenzyme or gene of a first family or species refers to distinct enzymesor genes of a second family or species which are determined byfunctional, structural or genomic analyses to be an enzyme or gene ofthe second family or species which corresponds to the original enzyme orgene of the first family or species. Most often, homologs will havefunctional, structural or genomic similarities. Techniques are known bywhich homologs of an enzyme or gene can readily be cloned using geneticprobes and PCR. Identity of cloned sequences as homolog can be confirmedusing functional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences).

As used herein, two proteins (or a region of the proteins) aresubstantially homologous when the amino acid sequences have at leastabout 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percentidentity of two amino acid sequences, or of two nucleic acid sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). In one embodiment, thelength of a reference sequence aligned for comparison purposes is atleast 30%, typically at least 40%, more typically at least 50%, evenmore typically at least 60%, and even more typically at least 70%, 80%,90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art (see,e.g., Pearson et al., 1994, hereby incorporated herein by reference).

In some instances “isozymes” can be used that carry out the samefunctional conversion/reaction, but which are so dissimilar in structurethat they are typically determined to not be “homologous”. For example,tktB is an isozyme of tktA, talA is an isozyme of talB and rpiB is anisozyme of rpiA.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). The followingsix groups each contain amino acids that are conservative substitutionsfor one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D),Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R),Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A),Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Sequence homology for polypeptides, which can also be referred to aspercent sequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild type protein and amutein thereof. See, e.g., GCG Version 6.1.

A typical algorithm used comparing a molecule sequence to a databasecontaining a large number of sequences from different organisms is thecomputer program BLAST (Altschul, 1990; Gish, 1993; Madden, 1996;Altschul, 1997; Zhang, 1997), especially blastp or tblastn (Altschul,1997). Typical parameters for BLASTp are: Expectation value: 10(default); Filter: seg (default); Cost to open a gap: 11 (default); Costto extend a gap: 1 (default); Max. alignments: 100 (default); Word size:11 (default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62.

When searching a database containing sequences from a large number ofdifferent organisms, it is typical to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using FASTA, a program in GCG Version 6.1.FASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences (Pearson,1990, hereby incorporated herein by reference). For example, percentsequence identity between amino acid sequences can be determined usingFASTA with its default parameters (a word size of 2 and the PAM250scoring matrix), as provided in GCG Version 6.1, hereby incorporatedherein by reference.

The disclosure provides accession numbers for various genes, homologsand variants useful in the generation of recombinant microorganismdescribed herein. It is to be understood that homologs and variantsdescribed herein are exemplary and non-limiting. Additional homologs,variants and sequences are available to those of skill in the art usingvarious databases including, for example, the National Center forBiotechnology Information (NCBI) access to which is available on theWorld-Wide-Web.

Culture conditions suitable for the growth and maintenance of arecombinant microorganism provided herein are described in the Examplesbelow. The skilled artisan will recognize that such conditions can bemodified to accommodate the requirements of each microorganism.Appropriate culture conditions useful in producing a acetyl-phosphate,acetyl-CoA or other metabolites derived therefrom including, but notlimited to 1-butanol, n-hexanol, 2-pentanone and/or octanol productscomprise conditions of culture medium pH, ionic strength, nutritivecontent, etc.; temperature; oxygen/CO₂/nitrogen content; humidity; lightand other culture conditions that permit production of the compound bythe host microorganism, i.e., by the metabolic action of themicroorganism. Appropriate culture conditions are well known formicroorganisms that can serve as host cells.

It is understood that a range of microorganisms can be modified toinclude a recombinant metabolic pathway suitable for the production ofn-butanol, n-hexanol and octanol. It is also understood that variousmicroorganisms can act as “sources” for genetic material encoding targetenzymes suitable for use in a recombinant microorganism provided herein.

The term “microorganism” includes prokaryotic and eukaryotic microbialspecies from the Domains Archaea, Bacteria and Eucarya, the latterincluding yeast and filamentous fungi, protozoa, algae, or higherProtista. The terms “microbial cells” and “microbes” are usedinterchangeably with the term microorganism.

The term “prokaryotes” is art recognized and refers to cells whichcontain no nucleus or other cell organelles. The prokaryotes aregenerally classified in one of two domains, the Bacteria and theArchaea. The definitive difference between organisms of the Archaea andBacteria domains is based on fundamental differences in the nucleotidebase sequence in the 16S ribosomal RNA.

The term “Archaea” refers to a categorization of organisms of thedivision Mendosicutes, typically found in unusual environments anddistinguished from the rest of the prokaryotes by several criteria,including the number of ribosomal proteins and the lack of muramic acidin cell walls. On the basis of ssrRNA analysis, the Archaea consist oftwo phylogenetically-distinct groups: Crenarchaeota and Euryarchaeota.On the basis of their physiology, the Archaea can be organized intothree types: methanogens (prokaryotes that produce methane); extremehalophiles (prokaryotes that live at very high concentrations of salt([NaCl]); and extreme (hyper) thermophilus (prokaryotes that live atvery high temperatures). Besides the unifying archaeal features thatdistinguish them from Bacteria (i.e., no murein in cell wall,ester-linked membrane lipids, etc.), these prokaryotes exhibit uniquestructural or biochemical attributes which adapt them to theirparticular habitats. The Crenarchaeota consists mainly ofhyperthermophilic sulfur-dependent prokaryotes and the Euryarchaeotacontains the methanogens and extreme halophiles.

“Bacteria”, or “eubacteria”, refers to a domain of prokaryoticorganisms. Bacteria include at least 11 distinct groups as follows: (1)Gram-positive (gram+) bacteria, of which there are two majorsubdivisions: (1) high G+C group (Actinomycetes, Mycobacteria,Micrococcus, others) (2) low G+C group (Bacillus, Clostridia,Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2)Proteobacteria, e.g., Purple photosynthetic+non-photosyntheticGram-negative bacteria (includes most “common” Gram-negative bacteria);(3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes andrelated species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7)Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria(also anaerobic phototrophs); (10) Radioresistant micrococci andrelatives; and (11) Thermotoga and Thermosipho thermophiles.

“Gram-negative bacteria” include cocci, nonenteric rods, and entericrods. The genera of Gram-negative bacteria include, for example,Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella,Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella,Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter,Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium,Chlamydia, Rickettsia, Treponema, and Fusobacterium.

“Gram positive bacteria” include cocci, nonsporulating rods, andsporulating rods. The genera of gram positive bacteria include, forexample, Actinomyces, Bacillus, Clostridium, Corynebacterium,Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus,Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

The disclosure includes recombinant microorganisms that comprise atleast one recombinant enzymes of the MEC pathway set forth in FIG. 1.For example, chemoautotrophs, photoautotroph, and cyanobacteria cancomprise native F/Xpk enzymes, accordingly, overexpressing FPK, XPK, orF/Xpk by tying expression to a non-native promoter can producemetabolite to drive the MEC pathway when combined with the otherappropriate enzymes of FIG. 1. Additional enzymes can be recombinantlyengineered to further optimize the metabolic flux, including, forexample, balancing ATP, NADH, NADPH and other cofactor utilization andproduction.

In another embodiment, a method of producing a recombinant microorganismthat comprises optimized carbon utilization including a MEC pathway toconvert methanol, methane or formaldehyde to acetyl-phosphate,acetyl-CoA or other metabolites derived therefrom including, but notlimited to, 1-butanol, 2-pentanone, isobutanol, n-hexanol and/or octanolis provided. The method includes transforming a microorganism with oneor more recombinant polynucleotides encoding polypeptides selected fromthe group consisting of a phosphoketolase (e.g., Fpk, Xpk, or Fpk/Xpk),a transaldolase (e.g., Tal), a transketolase (e.g., Tkt),ribose-5-phosphate isomerase (e.g., Rpi), a ribulose-5-phosphateepimerase (e.g., Rpe), a hexulose-6-phsophate synthase (e.g., Hps), ahexulose-6-phsophate isomerase (e.g., Phi), a dihydroxyacetone synthase(e.g., Das), a fructose-6-phosphate aldolase (e.g., Fsa), a methanoldehydrogenase (e.g., Mdh), a keto thiolase or acetyl-CoAacetyltransferase activity, hydroxybutyryl CoA dehydrogenase activity,crotonase activity, crotonyl-CoA reductase or butyryl-CoA dehydrogenaseactivity, trans-enoyl-CoA reductase and alcohol dehydrogenase activity.

In another embodiment, as mentioned previously, a recombinant organismas set forth in any of the embodiments above, is cultured underconditions to express any/all of the enzymatic polypeptide and theculture is then lysed or a cell free preparation is prepared having thenecessary enzymatic activity to carry out the pathway set forth in FIG.1 and/or the production of a 1-butanol, isobutanol, n-hexanol, octanol,2-pentanone among other products.

As previously discussed, general texts which describe molecularbiological techniques useful herein, including the use of vectors,promoters and many other relevant topics, include Berger and Kimmel,Guide to Molecular Cloning Techniques, Methods in Enzymology Volume 152,(Academic Press, Inc., San Diego, Calif.) (“Berger”); Sambrook et al.,Molecular Cloning—A Laboratory Manual, 2d ed., Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”) andCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (supplemented through 1999)(“Ausubel”), each of which is incorporated herein by reference in itsentirety.

Examples of protocols sufficient to direct persons of skill through invitro amplification methods, including the polymerase chain reaction(PCR), the ligase chain reaction (LCR), Q-replicase amplification andother RNA polymerase mediated techniques (e.g., NASBA), e.g., for theproduction of the homologous nucleic acids of the disclosure are foundin Berger, Sambrook, and Ausubel, as well as in Mullis et al. (1987)U.S. Pat. No. 4,683,202; Innis et al., eds. (1990) PCR Protocols: AGuide to Methods and Applications (Academic Press Inc. San Diego,Calif.) (“Innis”); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; TheJournal Of NIH Research (1991) 3: 81-94; Kwoh et al. (1989) Proc. Natl.Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Nat'l. Acad. Sci.USA 87: 1874; Lomell et al. (1989) J. Clin. Chem. 35: 1826; Landegren etal. (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4:560; Barringer et al. (1990) Gene89:117; and Sooknanan and Malek (1995) Biotechnology 13:563-564.

Improved methods for cloning in vitro amplified nucleic acids aredescribed in Wallace et al., U.S. Pat. No. 5,426,039.

Improved methods for amplifying large nucleic acids by PCR aresummarized in Cheng et al. (1994) Nature 369: 684-685 and the referencescited therein, in which PCR amplicons of up to 40 kb are generated. Oneof skill will appreciate that essentially any RNA can be converted intoa double stranded DNA suitable for restriction digestion, PCR expansionand sequencing using reverse transcriptase and a polymerase. See, e.g.,Ausubel, Sambrook and Berger, all supra.

The invention is illustrated in the following examples, which areprovided by way of illustration and are not intended to be limiting.

EXAMPLES

To construct an in vitro system, all the MEC enzymes were acquiredcommercially or purified by affinity chromatography, tested foractivity, and mixed together in a properly selected reaction buffer. AcPconcentration is measured using an end-point colorimetric hydroxamatemethod.

After demonstrating the feasibility of the pathway using in vitroenzymatic systems, the pathway was engineered into Escherichia coli.Xylose was used because it avoids the complication of variousglucose-mediated regulations, including the use of phosphotransferasesystem for transport. In order to engineer a preliminary pathway forxylose in E. coli, two enzymes were overexpressed: F/Xpk (encoded byf/xpk from Bifidobacterium adolescentis) and Fbp (encoded by E. colifbp). Other enzymes were natively expressed in E. coli under theexperimental conditions. The genes encoding these two enzymes werecloned on a high copy plasmid (pIB4) under the control of the PLlacO-1IPTG-inducible promoter. The plasmid was transformed into three E. colistrains: JCL16 [wild type], JCL166[ΔldhA, ΔadhE, Δfrd], and JCL 118[ΔldhA, ΔadhE, Δfrd, ΔpflB]. The latter two strains were used to avoidpathways competing. The expression of F/Xpk and Fbp was demonstrated byprotein electrophoresis and their activities were confirmed by enzymeassays. After an initial aerobic growth phase for cell growth, high celldensity cells were harvested and re-suspended in anaerobic minimalmedium with xylose at a final OD₆₀₀ of 9. Anaerobic conditions were usedto avoid the oxidation of acetate through the TCA cycle. HPLC was usedfor monitoring xylose consumption and organic acids formation. Thewild-type host (JCL16) produced a mixture of lactate, formate,succinate, and acetate from xylose, and the yield on acetate was quitelow at about 0.4 acetates produced per xylose consumed, indicating thatEMP and other fermentative pathways out-competed the synthetic pathway.By removing several fermentative pathways by the Δldh, ΔadhE, and Δfrdknockouts in JCL166, the yield was increased to 1.1 acetate/xyloseconsumed. After further deleting pflB in JCL118, the yield reach thehighest level of 2.2 acetates/xylose consumed, approaching thetheoretical maximum of 2.5 mole of acetate/mole of xylose. Somesuccinate remained, presumably due to succinate dehydrogenase left overfrom the aerobic growth phase.

One useful enzyme in the pathway is the irreversible Fpk/Xpk which cansplit F6P or xylulose-5-phosphate into AcP and E4P or G3P, respectively.This class of enzymes has been well-characterized in heterofermentativepathways from Lactobacillae and Bifidobacteria. In Lactobacillae,glucose is first oxidized and decarboxylated to form CO₂, reducingpower, and xylulose-5-phosphate, which is later split to AcP and G3P.Xpks have also been found in Clostridium acetobutylicum where up to 40%of xylose is degraded by the phosphoketolase pathway. Bifidobacteria,utilizes the Bifid Shunt, which oxidizes two glucoses into two lactatesand three acetates. This process yields increase the ATP yield to 2.5ATP/glucose. In both variants G3P continues through the oxidative EMPpathway to form pyruvate. Thus these pathways are still oxidative andare not able to directly convert glucose to three two-carbon compounds.For the pathway to function effectively, Fpk/Xpk and Fbp aresimultaneously expressed. However, since Fbp is a gluconeogenic enzyme,it is typically not active in the presence of glucose. Thus, althoughthese organisms have all the genes necessary for the pathway, it isunlikely that the pathway is functional in these organisms in thepresence of glucose.

Enzyme abbreviations and EC no. are listed in Table A.

TABLE A Enzyme abbreviations and EC numbers: Name Abbrev. EC # VerifiedSource F6P-Phosphoketolase 1a Fpk 4.1.2.22 B. adolescentis*X5P-Phosphoketolase 1b Xpk 4.1.2.9 L. plantarum Transaldolase 2 Tal2.2.1.2 E. Coli Transketolase 3 Tkt 2.2.1.1 E. Coli Triose PhosphateIsomerase 6 Tpi 5.3.1.1 E. Coli Fructose 1,6 8 Fbp 3.1.3.11 E. ColiBisphosphatase Fructose 1,6 bisphosphate 7 Fba 4.1.2.13 E. Coli AldolaseRibose-5-phosphate 4 Rpi 5.3.1.6 E. Coli isomerase Ribulose-3-phosphate5 Rpe 5.1.3.1 E. Coli epimerase Glucokinase Glk 2.7.1.2 E. ColiGlucose-6-phosphate Zwf 1.1.1.49 E. Coli Dehydrogenase Phopshoglucoseisomerase Pgi 5.3.1.9 E. Coli Acetate Kinase Ack 2.7.2.1 E. ColiHexulose-6-phosphate Hps 4.1.2.43 M. capsulatus synthase Hexulose 6phosphate Phi 5.3.1.27 M. Capsulatus isomerase Dihydroxyacetone synthaseDas 2.2.1.3 C. boindii (formaldehyde transketolase)Phosphotransacetylase Pta 2.3.1.8 E. Coli Methanol dehydrogenase Mdh1.1.99.37 B. Methanolicus

Thermodynamics of MEC Enzymes.

The change in standard Gibbs free energy (ΔrG′° in kJ/mol) for each stepwas calculated using eQuilibrator with pH=7.5 and ionic strength=0.2 Mto represent E. coli's cytosolic environment. All values were obtainedusing the difference of the standard Gibbs free energy of formationbetween the products and reactants. Since standard state is set at 1 Mfor all reactants (including water), some of the values do notcorrespond with experimentally verified data.

Combination of the Underlying F/Xpk Pathway with the Dihydroxyacetone(DHA) Pathway.

The F/Xpk pathway can be combined with the DHA pathway, which isanalogous to the RuMP pathway for assimilation of formaldehyde. Thepathways are shown in FIG. 1. This pathway includes the action of thegene fructose-6-phosphate aldolase (fsa) which has been characterizedfrom E. coli. Though the native activity of this enzyme was reported tohave a high K_(m), recent design approaches have improved affinitytowards DHA. The overall pathway from two methanol to ethanol isfavorable with a ΔrG′°=−68.2 kJ/mol.

Construction of In Vivo Pathways.

For the in vivo production of acetate from xylose, the plasmid pIB4 wasmade using pZE12 as the vector, F/Xpk from B. adolenscentis and Fbp fromE. coli (JCL16 gDNA). The strains JCL16, JCL166, and JCL118 wereconstructed (see, e.g., Int'l Patent Publication No. WO 2012/099934).This was done using the P1 phage transduction method with the Keiocollection as the template for single-gene knockouts. The strains JCL166and JCL118 were transformed with pIB4. Single colonies were grown in LBmedium overnight and inoculated into fresh LB+1% xylose culture the nextday. After reaching an OD=0.4-0.6, the strains were induced with 0.1 mMIPTG. After overnight induction, the cells were concentrated ten-foldand resuspended anaerobically in M9 1% xylose. A small portion of theinduced cells was extracted for HIS-tag purification to verify theactivity of F/Xpk and Fbp, and the rest was incubated anaerobicallyovernight for acetate production. The final mixture was spin down at14,000 rpm, and a diluted supernatant was run on HPLC to measure xyloseand organic acid concentration.

Phosphoketolase in Nature.

Phosphoketolase have been known to exist in many bacteria such asBifidobacteria for decades. Bifidobacteria make up a large portion ofthe beneficial flora in human's stomach, are used in the fermentation ofvarious foods from yogurt to kimchi, and are even sold in a dehydratedpill form. These bacteria contain a unique pathway that can fermentsugars to a mixture of lactate and acetate. By using the F6P/X5Pphosphoketolase enzyme, they are able to obtain more ATP than otherfermentative pathways at 2.5 ATP/glucose.

Certain embodiments of the invention have been described. It will beunderstood that various modifications may be made without departing fromthe spirit and scope of the invention. Other embodiments are within thescope of the following claims. Chemoautotrophs, photoautotroph,cyanobacteria overexpress FPK, XPK, tied to non-native promoter.

What is claimed is:
 1. A recombinant prokaryote or yeast microorganismexpressing enzymes that catalyze the conversion described in (i)-(xi),wherein at least one enzyme or the regulation of at least one enzymethat performs a conversion described in (i)-(xi) is heterologous to themicroorganism: (i) the production of acetyl-phosphate anderythrose-4-phosphate (E4P) from fructose-6-phosphate and/or theproduction of acetyl-phosphate and glyceraldehyde 3-phosphate (G3P) fromxylulose 5-phosphate; (ii) the reversible conversion offructose-6-phosphate and E4P to sedoheptulose 7-phosphate (S7P) and(G3P); (iii) the reversible conversion of S7P and G3P toribose-5-phosphate and xylulose-5-phosphate; (iv) the reversibleconversion of ribose-5-phosphate to ribulose-5-phosphate; (v) thereversible conversion of ribulose-5-phosphate to xylulose-5-phosphate;(vi) the reversible conversion of xylulose-5-phosphate and E4P tofructose-6-phosphate and glyceraldehyde-3-phosphate; (vii) theconversion of formaldehyde and ribulose-5-phosphate toD-arabino-3-Hexulose 6-phosphate; (viii) the reversible conversion ofD-arabino-3-Hexulose 6-phosphate to fructose-6-phosphate; (ix) theconversion of formaldehyde and xylulose-5-phosphate toglyceraldehyde-3-phosphate and dihydroxyacetone; (x) the conversion ofglyceraldehyde-3-phosphate and dihydroxyacetone to fructose-6-phosphate;and (xi) the conversion of methanol and a oxidized electron acceptor toformaldehyde and a reduced electron acceptor; wherein the microorganismproduces acetyl-phosphate, or compounds derived from acetyl-phosphateusing a carbon source selected from the group consisting of methanol,methane, and formaldehyde and any combination thereof.
 2. Therecombinant prokaryote or yeast microorganism of claim 1, wherein themicroorganism is derived from an E. coli microorganism.
 3. Therecombinant prokaryote or yeast microorganism of claim 2, wherein the E.coli is engineered to express a phosphoketolase.
 4. The recombinantprokaryote or yeast microorganism of claim 3, wherein thephosphoketolase is Fpk, Xpk or a bifunctional F/Xpk enzyme.
 5. Therecombinant prokaryote or yeast microorganism of claim 1, wherein themicroorganism is engineered to heterologously expresses one or more ofthe following enzymes: (a) a phosphoketolase (F/Xpk); (b) atransaldolase (Tal); (c) a transketolase (Tkt); (d) a ribose-5-phosphateisomerase (Rpi); (e) a ribulose-5-phosphate epimerase (Rpe); (f) amethanol dehydrogenase (Mdh); (g) a hexulose-6-phosphate synthase (Hps);(h) a hexulose-6-phosphate isomerase (Phi); (i) a dihydroxyacetonesynthase (Das); and (j) a fructose-6-phosphate aldolase (Fsa).
 6. Therecombinant prokaryote or yeast microorganism of claim 5, wherein themicroorganism is further engineered to express a phosphotransacetylase.7. The recombinant prokaryote or yeast microorganism of claim 5, whereinthe microorganism is engineered to express a phosphoketolase derivedfrom Bifidobacterium adolescentis.
 8. The recombinant prokaryote oryeast microorganism of claim 7, wherein the phosphoketolase comprises asequence that is at least 49% identical to SEQ ID NO:2 and hasphosphoketolase activity.
 9. The recombinant prokaryote or yeastmicroorganism of claim 5, wherein the microorganism is engineered toexpress or over express a hexulose-6-phosphate synthase.
 10. Therecombinant prokaryote or yeast microorganism of claim 5, wherein themicroorganism is engineered to express or over express ahexulose-6-phosphate isomerase.
 11. The recombinant prokaryote or yeastmicroorganism of claim 5, wherein the microorganism is engineered toexpress or over express a dihydroxyacetone synthase.
 12. The recombinantprokaryote or yeast microorganism of claim 5, wherein the microorganismis engineered to express or over express a fructose-6-phosphatealdolase.
 13. The recombinant prokaryote or yeast microorganism of claim1, wherein the microorganism expresses enzymes selected from the groupconsisting of: (a) a phosphoketolase having at least 49% identity to SEQID NO:2; (b) a transaldolase having at least 30% identity to SEQ IDNO:10; (c) a transketolase having at least 41% identity to SEQ ID NO:12;(d) a ribose-5-phosphate isomerase having at least 37% identity to SEQID NO:8; (e) a ribulose-5-phosphate epimerase having at least 51%identity to SEQ ID NO:6; (f) a methanol dehydrogenase having at least95% identity to SEQ ID NO:4; (g) a hexulose-6-phosphate synthase havingat least 95% identity to SEQ ID NO:14; (h) a hexulose-6-phosphateisomerase having at least 95% identity to SEQ ID NO:16; (i) adihydroxyacetone synthase having at least 95% identity to SEQ ID NO:18;(j) a fructose-6-phosphate aldolase having at least 95% identity to SEQID NO:20; and (k) any combination of (a)-(j).